Discveory of SERCA1 specific small molecule inhibotirs based on the survival mechanisms of metastatic hepatocellular carcinoma cells dependent on CaMK2α-Mediated SERCA1 expression

Discveory of SERCA1 specific small molecule inhibotirs based on the survival mechanisms of metastatic hepatocellular carcinoma cells dependent on CaMK2α-Mediated SERCA1 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discveory of SERCA1 specific small molecule inhibotirs based on the survival mechanisms of metastatic hepatocellular carcinoma cells dependent on CaMK2α-Mediated SERCA1 expression Jin Hong Lim, Keunwan Park, Kyung Hwa Choi, JungMin Kim, Yoo-Lim Jhe, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7382767/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Refractory hepatocellular carcinoma (HCC) perpetuates metastasis or recurrence through anti-cancer drug resistance, necessitating more effective and reliable therapeutic strategies. Methods We propose a new therapeutic approach involving the discovery of novel small molecules through target identification and validation in a patient-derived metastatic HCC model. Results We showed that calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α)-mediated enhancement of sarco/endoplasmic reticulum (ER) calcium ATPase 1 (SERCA1) expression level was pivotal events under anti-cancer drug treated conditions in patient-derived metastatic HCC cells. Increased SERCA1 was regulates to overloaded free calcium. SERCA is widely recognized as a key regulator of cytosolic free calcium under severe ER stress conditions. However, a cardiac dysfunction was inevitable in vivo because of non-specific inhibition of SERCA isoforms by conventional SERCA inhibitors. Based on the molecular structure of SERCA1, we discovered and synthesized two SERCA1-specific inhibitors, candidate 56 and 62. These compounds significantly reduced tumor size in the metastatic HCC xenograft tumor model without cardiac contractile dysfunction. Conclusions This study first showed survival mechanism of patient-derived metastatic HCC cell, and propose a new therapeutic approach by the new small molecules, candidate 56 and 62, which are SERCA1 isoform-specific inhibitors without cardiac dysfunction by SERCA1 selectively inhibition. patient-derived metastatic HCC sarcoplasmic/endoplasmic reticulum calcium ATPase calcium/calmodulin-dependent protein kinase 2 alpha Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hepatocellular carcinoma (HCC) is a well-known form of liver cancer, comprising over 90% of hepatic carcinoma cases. Fortunately, advancements in systemic treatments for advanced HCC have led to a steady increase in patient survival rates[ 1 ]. However, the emergence of anti-cancer drug resistance in some cases presents a critical challenge, leading to patient mortality due to cancer recurrence and metastasis[ 2 ]. Molecular variations suggest the poor prognosis of HCC in patients with metastasis or recurrence; however, the fundamental mechanisms remain unclear. Therefore, more efficient and reliable therapeutic approaches are required. Recently, the ratio of relapse to survival after surgery was determined following systemic chemotherapy before surgery, regardless of cancer development[ 3 ]. Systemic chemotherapy is typically administered to patients with advanced cancer subtypes characterized by invasion or metastatic lesions, as local therapies are often inadequate against chemotherapy-resistant cancer[ 4 , 5 ]. Anti-cancer drug treatments exert significant stress on cancer cells, contributing to cellular viability challenges. Cancer stem cells (CSCs) have demonstrated resilience against metabolic stresses within tumor microenvironments through epigenetic reprogramming, emerging as a notable therapeutic target[ 6 ]. Under anti-cancer drug treatment conditions, CSCs exhibit enhanced survival mechanisms against acute sarco/endoplasmic reticulum (ER) stress compared to non-CSCs, prompting the exploration of various molecular strategies for epigenetic reprogramming[ 7 ]. Epigenetic reprogramming stimulated by anti-cancer drugs may involve several molecular regulators[ 8 , 9 ]. Previous studies have established calcium/calmodulin-dependent protein kinase alpha (CaMK2α) as a pivotal transcriptional regulator that inhibits cytosolic free calcium-mediated apoptosis by upregulating sarco/ER calcium ATPase (SERCA) levels[ 9 ]. Furthermore, CaMK was a key transcriptional regulator of increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression, which is a potent inducer of ATP production via mitochondrial respiration [ 10 ]. Cytosolic free calcium levels play a critical role in cellular responses to ER stress and subsequent cell fate decisions[ 11 ]. ER stress induces the release of cytosolic free calcium from the ER to the cytosol via IP3R (inositol 1,4,5-trisphosphate) receptors, which is regulated by calcium pumps, exchangers, and channels to maintain cellular calcium homeostasis[ 12 ]. Excessive elevation of cytosolic free calcium beyond physiological levels triggers apoptotic signals[ 13 ]. SERCA serves as a key regulator in managing overloaded cytosolic free calcium levels in cancer cells, contributing to cellular defense by efficiently restoring cytosolic free calcium to the ER[ 14 – 16 ]. Previously, we showed potential implications for applying new combinatorial strategies and discovering anti-cancer candidates that SERCA-targeted a specific vulnerability of anti-cancer drug-resistant-mediated cells[ 17 ]. However, a cardiac dysfunction was inevitable in xenograft model because of non-specific inhibition of SERCA isoforms by several candidates[ 17 , 18 ]. Injured myocardial calcium cycling is a key regulator of heart failure (cardiac dysfunction), causing to a change in the structure remodelling and contractile function of the heart[ 19 ]. In cardiomyocytes, the regulation of cytosolic free calcium storage and release by sarcoplasmic reticulum (SR) is mostly dependent on calcium regulation proteins, such as SERCA2a. For the relaxation phase of the cardiac cycle, SERCA2a is a pivotal regulator in transporting cytosolic free calcium back to the SR, to restore cytosolic free calcium levels to their resting state and replenish SR calcium levels for the next contraction. However, functional inhibition of SERCA2a by thapsigargin (non SERCA isoform specific inhibitor) causes to cardiac dysfunction[ 20 ]. The current study aimed to design a therapeutic approach with decrease of cardiac dysfunction based on CaMK2α-mediated enhancement of SERCA1 via the regulation of cytosolic free calcium levels. We further aimed to identify targets and novel small molecules for isoform-specific inhibition of metastatic HCC under acute ER stress in patient-derived samples. The findings of this study are clinically significant for the development of new small molecules with decrease of side effects that selectively and effectively target highly malignant cancer cells, including cancer stem-like cells and drug-resistant cancer cells. Materials and methods Patient characteristics The patients recruited in this study underwent liver resection. They were treated between January 2018 and December 2022 at the Severance Hospital (Seoul, South Korea). Details of patient presentations, surgical and pathological findings, and outcomes were obtained from the Cancer Center Database. All procedures involving patients were performed in accordance with institutional ethical standards, applicable local/national regulations, and guidelines of the 1964 Helsinki Declaration and its later amendments. Further details are provided in Supplementary Methods. Patient tissue specimens Tumor specimens were obtained from patients with biochemically and histologically confirmed recurrent or metastatic tumors who were treated at Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Fresh tumor specimens were obtained for surgical resection. Ethical considerations The study protocol was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (Institutional Review Board Protocol: 3-2022-0331). Samples were obtained from patients at Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea. The requirement for obtaining formal written consent was waived owing to the retrospective observational nature of the study. Further patient details are provided in the Supplementary Methods. Tumor cell isolation and primary culture After resection, the tumors were kept in normal saline containing antifungal agents and antibiotics and moved to the laboratory. Normal tissues and fat were removed, and the tissues were rinsed with 1× Hank’s Balanced Salt Solution. The cells were resuspended in Rosewell Park Memorial Institute (RPMI)-1640 medium (Hyclone, South Logan, UT, USA) with 20% FBS (Hyclone) and 2% penicillin/streptomycin solution (Gibco, Grand Island, NY, USA). Further details of the protocol have been Supplementary Methods or previously published[ 8 ]. Cell culture The patient-derived cancer cells, THLE-2, Huh-7, HepG2 and Hep3B cells were obtained from fresh tumors of patients or ATCC (ATCC, Manassas, VA, USA). YUMC-NM-H1 (non-metastatic HCC) and YUMC-M-H1, -H2, and -H3 (metastatic HCC) cells were isolated from patients with HCC treated at the Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Cells were grown in RPMI-1640 medium containing 20% FBS. Cells were authenticated using short tandem repeat profiling, karyotyping, and isoenzyme analysis. Mycoplasma contamination was evaluated using a Lookout Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, MO, USA; MP0035). mRNA sequencing and analysis Whole RNA was isolated from the patient-derived cancer cells, YUMC-NM-H1 (non-metastatic HCC) and YUMC-M-H1, -H2, and –H3 (metastatic HCC), using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Paired-end sequencing reads were generated on the Illumina sequencing NovaSeqX platform. Before starting analysis, Trimmomatic v0.38 was used to remove adapter sequences and trim bases with poor base quality. RNA enrichment (total RNA) in current study, we preprocessed the raw reads from the sequencer to remove low quality and adapter sequences before analysis and aligned the processed reads to the Homo sapiens genome assembly (GRCh38) using HISAT v2.1.0 (HISAT2, RRID: SCR 015530)[ 21 ]. HISAT utilizes two types of indexes for alignment: a global, whole-genome index, and tens of thousands of small local indexes. Both are constructed using the same Burrows–Wheeler transform (BWT) or graph FM index (GFM) as Bowtie2 (Bowtie 2, RRID: SCR 016368). The reference genome sequence of Homo sapiens (GRCh38) and annotation data were downloaded from the National Center for Biotechnology Information (NCBI). Aligned data (SAM file format) were sorted and indexed using SAMtools v 1.9. After alignment, transcript assembly of known transcripts was processed using StringTie v2.1.3b (StringTie, RRID: SCR016323)[ 22 ]. Based on these results, expression abundance of transcript and gene were calculated as read count or fragments per kilobase of exon per million fragments mapped (FPKM) value per sample. Statistical analysis of gene expression level The relative abundances of genes were measured in Read Count using StringTie. We performed statistical analyses to find differentially expressed genes using the estimates of abundances for each gene in the samples. Genes with more than one Read Count values in the samples were excluded. To facilitate log2 transformation, 1 was added to each Read Count value of filtered genes. Filtered data were log2-transformed and subjected to trimmed mean of M-values (TMM) normalization. The statistical significance of the differential expression data was determined using exactTest, edgeR and fold change, in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting the p-value using the Benjamini-Hochberg algorithm. However statistical analysis in current study was used raw p-value instead to FDR. Because if used FDR, not acquired sufficiently gene list for analysis of enrichment for GO and KEGG. For differentially expressed genes (DEGs) sets, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-enrichment and functional annotation analysis and pathway analysis for significant gene list were performed based on Gene Ontology and KEGG pathway analyses. siRNA transfection for CaMK2α knockdown Patient-derived metastatic HCC cells, YUMC-M-H1, -H2, and -H3 cells were transfected with CaMK2α siRNA or scrambled control siRNA. Sequence of the CaMK2α siRNA was designed using si-Designed software (Bioneer, Daejeon, South Korea) and the siRNA duplex was purchased from Bioneer. The sense and antisense sequences of the siRNA were as follows: 5'-UGAUCGAA GCCAUAAGCAA(dTdT)-3' (forward) and 5'-UUGCUUAUGGCUUCGAUCA(dTdT)-3' (reverse). Further details are provided in our previously published article[ 9 ]. Subcellular fractionation and immunoblot analysis Primary antibodies against GPC3 (1:500, Cat#174851, Abcam, Cambridge, UK), HSP70 (1:500, Cat#2787, Abcam), TP (1:500, Cat#226917, Abcam), SERCA1 (1:200, Cat#133275, Abcam), SERCA2 (1:400, Cat#137020, Abcam), SERCA3 (1:200, Cat#154259, Abcam), Bcl-2 (1:400, Cat#196495, Abcam), pCaMK2α (1:500, Cat#171095, Abcam), CaMK2α (1:500, Cat#92332, Abcam), pIKKα (1:500, Cat#138426, Abcam), CHOP (1:500, Cat#7351, Santa Cruz Biotechnology, CA, USA), pNFκB (1:500, Cat#86299, Abcam), Histone H3 (1:500, Cat#18521, Abcam), caspase-3 (1:500, Cat#9661, Cell Signaling Technology, Beverly, MA,USA) and β-actin (1:500, Cat#47778) were used. Equal amounts of protein were separated on an 8–10% sodium dodecyl sulfate-polyacrylamide gel, and the resolved proteins were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were subsequently blocked with 5–10% nonfat milk in TBST for 1 h at room temperature or overnight at 4°C and incubated with appropriate concentrations of primary antibodies overnight at 4°C. The membranes were then rinsed 3–10 times with TBST and probed with the corresponding secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) at room temperature for 1 h. After rinsing, the blots were developed with ECL reagents (Pierce) and exposed to Kodak X-OMAT AR Film (Eastman Kodak, Rochester, NY, USA) for 1–5 min. Nuclear fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific; #78833) in accordance with the manufacturer's instructions. Separated nuclear and cytoplasmic extracts were isolated using a protein extraction solution (PRO-PREP, iNtRON Biotechnology, Seoul, Korea, #17081) or a histone extraction kit (Abcam, Cambridge, UK, #113476). Protein extracts and bands were quantified using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and the ImageJ software (NIH, Bethesda, MD, USA). Further details are provided in the Supplementary Methods. Electrophoretic mobility shift assay (EMSA) The DNA binding activity of NFκB against the SERCA1 promoters was confirmed using a 32 P-labelled oligonucleotide. Specific labelled and unlabelled oligonucleotides are as follows. The DNA binding activity of NFκB against the SERCA1 promoter was confirmed using a 32 P-labelled oligonucleotide. Labelled and unlabelled oligonucleotides specific for NFκB (Forward 5′-GGGGGGTTCCC-3′, Reverse 5′-GGGAACCCCCC-3′) and mutant NFκB (Forward 5′-GGGcGcTTCCC-3′, Reverse 5′-GGGAAgCgCCC-3′) were synthesized by Bioneer. Further protocol details are as described in Supplementary Methods. Dual Luciferase Reporter Assay Promoter activity was evaluated using the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA; E1960), according to the manufacturer's protocol. Regions of NFκB binding sites were amplified by PCR from human genomic DNA (NFκB primers: Forward 5′-GGGGGGTTCCC-3′, Reverse 5′-GGGAACCCCCC-3′). The PCR products were cloned into the pGL4.70 promoter Vector (Promega) using T4 DNA ligase (Thermo Scientific, Waltham, MA, USA; EL0011). All insertions were confirmed by sequencing. Further protocol details are as described in Supplementary Methods. Immunofluorescence analysis and confocal imaging The expression level of nuclear translocated pNFκB was analyzed by immunofluorescence staining. Cells cultured on glass-bottomed dishes (MatTek, Ashland, MA. USA) were fixed with 4% formaldehyde solution (R&D Systems, Abingdon, UK) for 10 min and permeabilized with 0.5% TritonX-100 in phosphate-buffered saline (PBS) for 10 min. The slides were air dried, washed with PBS, and incubated overnight at 4°C with anti-pNFκB (1:50, Cat#86299, Abcam, Cambridge, UK) in 3% bovine serum albumin included PBS. Further details are provided in the Supplementary Methods. Cytosolic free calcium measurements using microspectrofluorimetry Cytosolic free calcium levels in patient-derived HCC cells were imaged using a calcium-sensitive fluorescent dye, Fura-2AM. HCC cells were incubated with Fura-2AM in normal phosphate buffered saline solution for 30 min at 37 ℃, followed by de-esterification of the dye for another 30 min at 22–25 ℃. Further details are provided in a previously published article[ 8 ]. Cell viability assay Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were seeded in 96-well plates at a density of 6 × 10 3 cells per well and cultured overnight to achieve 80% confluency. Further details on the protocol are available in existing publications[ 9 , 23 ]. The assay was performed thrice and cell viability was evaluated as a percentage of the signal observed in vehicle-treated cells and reported as the means ± standard error of the mean. Molecular docking simulation for SERCA1 The three-dimensional structure of SERCA1 was prepared using AlphaFoldDB[ 24 ] with the UniProt ID O14983. Most of the residues in the alpha-fold-predicted structure had high confidence scores, confirming reliability of the prediction model. The modeled human SERCA1 structure was further refined using Rosetta Relax[ 25 ] for use in subsequent docking simulations to elucidate crucial ligand-receptor interactions. The docking simulations for the two active compounds were conducted using DiffDock, which exhibits superior performance in blind docking tasks (i.e., docking simulation without knowing prior binding site information) The docked complex model was refined using RosettaLigand by performing iterative local docking refinement and energy minimization[ 26 ]. The final docking model was selected based on the interface binding energy (interface delta score) and used to analyze detailed molecular interactions. Total RNA extraction and quantitative reverse transcription-polymerase chain reaction Total RNA was extracted from tumor cells using the RNeasy Mini Kit (Qiagen, cat. 74106) and One-Step reverse transcription-polymerase chain reaction (RT-PCR) Kit (Qiagen, Cat#204243) according to the manufacturer’s protocols. All data were normalized to α-tubulin expression. The following primers for SERCA1, SERCA2 , and SERCA3 were used for quantitative RT-PCR (qRT-PCR) analysis: SERCA1 , 5'-GTGATCCGCCAGCTAATG-3' (forward) and 5'-CGAATGTCAGGTCCGTCT-3' (reverse); SERCA2 , 5'-GGTGGTTCATTGCTGCTGAC-3' (forward) and 5'-TTTCGGACAAGCTGTT GAGG-3' (reverse); SERCA3 , 5'-GATGGAGTGAACGACGCA-3' (forward) and 5'-CCA GGTATCGGAAGAAGAG-3' (reverse); and α-tubulin; 5'- CGGGCAGTGTTTGTAGACTTGG-3' (forward) and 5'-CTCCTTGCCAATGGTGTAGTGC-3' (reverse). In vivo mouse xenograft model with patient-derived HCC cells All the experiments were performed in accordance with the guidelines established by the Animal Experimentation Committee of Yonsei University. Patient-derived HCC cells (4.5 × 10 6 cells/mouse) were injected subcutaneously into the upper left flank region of female NOD/Shi-scid, IL-2Rγ KOJic (NOG) mice. Animals were maintained under specific pathogen-free conditions. All experiments were approved by the Animal Experiment Committee of Yonsei University (IACUC approval No 2022 − 0105). The detailed protocol is described in the Supplementary Methods. Masson trichrome and H&E (Hematoxylin and Eosin) staining for establishment to hepatic and cardiac injury Hepatic and cardiac biopsy specimens fixed in 10% buffered formalin solution were embedded with paraffin. Sections (5 µm) were stained with masson trichrome or H&E. The degree of cellular changes in hepatic and cardiac injury was evaluated with masson trichrome or H&E staining according to the protocol provided by the manufacturer protocol. The procedure stains nuclei dark red, cytoplasm and muscle fibres red, and ECM components blue. Statistical analyses Statistical analyses were performed using GraphPad Prism (version 6.0; GraphPad Software, La Jolla, CA, USA), and immunohistochemistry results were evaluated using analysis of variance, followed by the Bonferroni post-hoc test. Values are expressed as the mean ± standard deviation, and p < 0.05 indicated statistical significance. Results Patients and disease characteristics This study targeted and isolated cancer cell from speciment of cancer patients who took anti-cancer drugs but caused metastasis. The patient group consists of nine men and five women, a total of fourteen (Supplementary Fig. 1A and Table 1 ). Compared to patient-derived non-metastatic (included Huh7) or metastatic HCC cells, metastatic HCC cells showed increase of metastatic markers ( NDUFA4L2, EBF1, TYMP [Thymidine phosphorylase] and MET ) (Supplementary Fig. 1B), cancer stemness markers ( PROM1 [CD133], ALDH1A1, KRT17 and KRT19 ) (Supplementary Fig. 1C) and EMT markers ( SNAIL1 and 2, ZEB 1 and 2, WNT1 and 4, TWIST1 ) (Supplementary Fig. 1D) than non-metastatic HCC. Well-known HCC marker GPC-3 (glypican-3) and HCP70 (Heat shock protein 70) expression was compared normal liver cell (THLE-2) and established HCC cell line (Huh7, HepG2, Hep3B) in patient-derived HCC cells (Supplementary Fig. 1E). Among the 14 patient, 3 patient-derived HCC cells were selected for current study. The patient cohort comprised four men (Table 1 and Supplementary Fig. 2). Detailed characteristics of patient-derived hepatocellular carcinoma (HCC) and clinical features are provided in Table 1 . The non metastatic HCC cell line, YUMC-NM-H1, was isolated from the postoperative specimen of a 71-year-old woman who was unexposed to preoperative chemotherapy or radiotherapy (Supplementary Fig. 2A and E). Conversely, metastatic cell lines, YUMC-M-H1, YUMC-M-H2, and YUMC-M-H3, were isolated from specimens of three patients (53-year-old woman, 67 and 71-year-old man) with metastasis HCC at the time of surgery (Supplementary Fig. 2B–D and F–H); among these, two patients had peritoneal and diaphragmatic metastases. The primary tumors at the time of surgery and preoperative radiologic findings are presented in Supplementary Fig. 2. In the computed tomography (CT) findings, the yellow circle (Supplementary Fig. 2A) indicates a primary mass without metastasis (Supplementary Fig. 2E) and red circles (Supplementary Fig. 2B–D) indicate metastatic HCC (Supplementary Fig. 2F–H). Differential gene expression and signal activation between non-metastatic and metastatic HCC cells in sorafenib treatment-induced acute ER stress conditions Previously, we demonstrated that acute glucose deprivation or anti-cancer drug treatment increases metabolic stress, leading to favorable subclone classification with CSC properties[ 8 , 9 ]. Furthermore, under acute ER stress conditions, CSC-like properties and intensified induction of signaling pathways involved in survival are observed in some selectively surviving cells compared to those in their parental lineages[ 8 , 9 ]. To identify differences in gene expressions and signaling pathways between non-metastatic and metastatic HCC cells under anti-cancer drug-induced acute ER stress, we performed RNA sequencing-mediated transcriptome analysis. Current study, we used the patient-derived HCC cells, YUMC-NM-H1 (non-metastatic HCC) and YUMC-M-H1, -H2, and -H3 (metastatic HCC). Four tissue samples (patients were treated at the Severance Hospital, Yonsei University College of Medicine, Seoul, Korea) were obtained from two patients with divergent HCC properties (Table 1 , HCC in non-metastatic HCC was termed “YUMC-NM-H1” and that in metastatic as “YUMC-M-H1, -H2, and -H3”). Considering that gene expression is primarily dependent on metastasis, we hypothesized that metastasis involves an advanced cancer phenotype induced by epigenetic reprogramming, relative to that in non metastatic HCC cells. Therefore, metastatic HCC cell survival may involve epigenetic gene pathway induction which regulates calcium (for overburdened cytosolic free calcium regulation), peroxisome proliferator-activated receptors (PPAR; for mitochondrial metabolism), nuclear factor kappa B (NFκB; for metabolic adaptation or energy homeostasis), and Notch and Wnt signaling pathways under anti-cancer drug-treated acute ER stress conditions. NFκB is an anti-apoptotic factor known for its transcriptional role in SERCA expression[ 9 ]. Moreover, NFκB is a pivotal regulator of mitochondrial respiration, metabolic adaptation, and energy homeostasis[ 27 ]. Compared to non-metastatic HCC cells (YUMC-NM-H1), metastatic markers ( TYMP [Thymidine phosphorylase], EBF1 [early B cell factor 1], NDUFA4L2 [NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2], and MET [Mesenchymal epithelial transition factor] ), multidrug resistance related gene (ABCC1 [MRP]), cancer stemness markers ( PROM1 [CD133], ALDH1A1, KRT17, KRT19, CD44, CD24 and SOX2 ) and EMT markers ( SNAIL1, SNAIL2, WNT1 WNT4, ZEB1[Zinc finger E-box-binding homeobox1], ZEB2 and TWIST1[twist family bHLH transcription factor1] ) were significantly increased in metastatic HCC cells, YUMC-M-H1, -H2 and -H3 (Fig. 1 A and 1 E–H). In metastatic HCC, we concentrated on highly ranked gene modules involved in survival-related signaling pathways, including Notch, calcium, Hedgehog, and Wnt (Fig. 1 B), particularly calcium signaling pathways which were overstimulated in metastatic HCC cells. Well-known HCC marker (GPC-3 and HCP70) and metastatic marker (TP; Thymidine phosphorylase) expression was compared normal liver cell (THLE-2) and established HCC cell line (Huh7, HepG2, Hep3B) in patient-derived HCC cells (Fig. 1 C). Basal level of HCC and metastatic marker expressions were significantly increased in established HCC and patient-derived metastatic HCC than normal liver cell (Fig. 1 C). Basal SERCA1 and anti-apoptosis marker Bcl-2 (B cell lymphoma-2) levels (critical components involved in anti-apoptosis and calcium homeostasis)[ 28 ] were significantly higher in metastatic HCC cells than in non-metastatic HCC cells. On treatment with anti-cancer drugs such as sorafenib, SERCA1 and Bcl-2 expression notably increased in metastatic HCC cells compared to those in non-metastatic HCC cells (Fig. 1 D). SERCA2 and SERCA3 expression showed no significant differences between the two HCC cells, regardless of sorafenib treatment (Fig. 1 D). In summary, the calcium regulation signaling pathway or survival-related gene simulation in managing calcium regulation and SERCA are pivotal factors for survival under anti-cancer drug, sorafenib-induced metabolic stress conditions in metastatic HCC cells. Metastatic HCC cell survival during prolonged anti-cancer drug treatment-stimulated metabolic stress depends on increased CaMK2α-mediated SERCA1 expression Excessive cytosolic free calcium is primarily involved in cell death[ 29 ]. Calcium channels, exchangers, and pumps are managed by a cytosolic-free calcium overburden and are pivotal regulators of cellular fate in physiological and pathological microenvironments[ 12 , 29 ]. CaMK2α is a known as factor to increase in the nuclear translocation of NFκB which regulates of Bcl-2, SERCA and PGC1α [ 10 , 28 , 30 ]. We initially determined alterations in pCaMK2α, pIKKα (I kappa B kinase), Bcl-2 (anti-apoptotic marker), SERCA1, CHOP ( C/EBP homologous protein , ER stress marker), cleaved-caspase3 (apoptotic marker), nuclear located NFκB in the presence or absence of anti-cancer drug (sorafenib) in non-metastatic (YUMC-NM-H1) and metastatic (YUMC-M-H1, -H2 and -H3) HCC (Fig. 2 A). Under anti-cancer drug treatment-mediated acute ER stress conditions, metastatic HCC was evaded ER stress (CHOP) or apoptosis (cleaved-caspase3) via increase of nuclear located NFκB, Bcl-2 and SERCA1 by CaMK2α (Fig. 2 A). Based on results of electrophoretic mobility shift aasy (EMSA), enhancement the nuclear translocation of NFκB (Supplementary Fig. 3A–C) by stimulating CaMK2α (pCaMK2α, phosphorylated CaMK2α), which subsequently upregulated the transcription of SERCA1 and Bcl-2 in metastatic HCC cells compared to that in non-metastatic HCC cells (Fig. 2 A and Supplementary Fig. 3A–C). Consequently, we evaluated the causal relationship between elevated SERCA1 levels and CaMK2α by CaMK2α knockdown in metastatic HCC (YUMC-M-H1, -H2, and -H3 small interfering [si]- CaMK2α ) with sorafenib (Fig. 2 B–E). Immunoblot analysis (Fig. 2 B) and cell viability assay (Fig. 2 C) indicated that CaMK2α knockdown in metastatic HCC cells using siRNAs downregulated cell viability and decrease of pIKKα, Bcl-2 and SERCA1 levels through inhibition of nuclear translocated NFκB under sorafenib treatment conditions (Fig. 2 B–C). Consequently, CaMK2α knockdown in metastatic HCC cells significantly increased the levels of apoptotic markers CHOP and cleaved caspase 3, while considerably decreasing those of pCaMK2α, pIKKα, Bcl-2, SERCA1, and nuclear translocated NFκB under acute ER stress conditions by sorafenib treatment (Fig. 2 B). Viability assays showed the dose-dependent restriction of prolonged survival with sorafenib in downregulated CaMK2α metastatic HCC cells (Fig. 2 C, left; YUMC-M-H1, middle; YUMC-M-H2, right; YUMC-M-H3). Immunofluorescence assays of CaMK2α knockdown in metastatic HCC cells showed that the nuclear translocation of NFκB was considerably inhibited by CaMK2α knockdown (Fig. 2 D). Microspectrofluorimetry revealed that the CaMK2α knockdown-mediated inhibition of SERCA1 and Bcl-2 influenced intracellular calcium levels in metastatic HCC cells following sorafenib treatment (Fig. 2 E). Cytosolic free calcium levels were measured in response to high potassium depolarization, which triggered an increase in the levels of cytosolic free calcium; its clearance was estimated in the presence or absence of sorafenib based on separate calcium fluxes. Cytosolic free calcium levels were restored to the basal levels in the control and si-scrambled group after sorafenib each treatment in metastatic HCC cells (Fig. 2 F, top; YUMC-M-H1, middle; YUMC-M-H2 and bottom; YUMC-M-H3). However, this restoration was interrupted by CaMK2α knockdown via si -CaMK2α in metastatic HCC cells. In summary, metastatic HCC cells can ameliorate calcium-mediated apoptosis under anti-cancer drug treatment by increasing SERCA1 levels through inducing nuclear NFκB translocation via CaMK2α, which is a pivotal transcriptional contributor to SERCA1 and Bcl-2 upregulation. Candidate 56 and 62 identification as SERCA1 target-specific inhibitors using molecular docking simulation and structure modeling We hypothesized and demonstrated that metastatic HCC cells and functional SERCA1 inhibition could be a practical clinical approach for patients with metastatic HCC. We identified novel, small SERCA1-specific binding molecules as well as the possibility of pharmacophore-binding modes. Consequently, we identified two novel small molecules, candidate 56 and 62, which showed pharmacophoric high SERCA1-specific binding affinity, resulting in critical functional suppression of SERCA1. An evolutionary chemical binding similarity (ECBS) program was used to elucidate the inhibitory mechanisms of candidate 56 and 62 (Fig. 3 A–C). ECBS method using a categorization similarity learning framework defined with paired chemical data and target’s evolutionary relationship. The ECBS method is designed to encode molecular features enriched in evolutionarily conserved chemical-target binding relationships, and formulated by the likelihood of chemical compounds binding to identical targets[ 31 ]. To recognize the molecular interactions of candidate 56 and 62, we carried out blind docking simulations, followed by local docking refinement and energy minimization. A blind docking simulation was performed using the human SERCA1 structural model to predict the ligand-binding site and molecular interactions (Fig. 3 D and G). A similarity search using FoldSeek[ 32 ] server revealed that the structure of human SERCA1 is highly similar to that of rabbit SERCA1 (PDB ID 6YSO, UniProt ID P04191), with a sequence identity of 95.3% and root mean square deviation of 1.89 Å (TM-Score 0.971), indicating significant consistency between them. Docking complex models suggested the potential binding of candidate 56 and 62 to the cavity between the n and p domains in the cytoplasmic region of SERCA1, overlapping with the binding site of the ATP derivative (PubChem CID 644358) found in the rabbit SERCA1 structure[ 33 ]. The docking scores, obtained through all-atom minimization with Rosetta, were − 14.3 and − 13.4 (in Rosetta Energy Unit) for candidate 56 and 62, respectively. Despite their similar binding scores and chemical structures, candidate 56 and 62 exhibited contrasting binding orientations in the docking models, presumably due to their distinct 3D conformations (Fig. 3 E and F). In candidate 56, the aromatic ring with a chlorine atom was directed towards F487 and M494; whereas in candidate 62, it interacted with P518 (Fig. 3 E and F). Notably, F487 consistently participated in pi-pi interactions with the aromatic rings of both compounds. Additionally, R560 showed a polar interaction only with candidate 62, presumably contributing to its binding specificity, along with additional interactions with L562 and T441. In contrast, A517 and K492 cells exhibited more hydrophobic interactions with candidate 56. Pharmacophore models based on the receptor–ligand interactions are constructed to highlight the essential interactions within the ligands (Fig. 3 H). These ligand–protein interactions observed in the docking models have the potential to offer valuable insights into the molecular basis of their activity, aiding efficient molecule design in the future. These protein-ligand interactions, as suggested by the docking models, have the potential to elucidate the molecular basis of the ligand activity. However, experimental validation is still necessary in future studies. Candidate 56 and 62 increase of restraint the survival of metastatic HCC cells through failed to revert to basal levels after cytosolic free calcium spike We conducted cell viability assays to assess the anti-cancer impact of the novel small molecules and SERCA1-specific inhibitors, candidate 56 and 62. The results showed that non-metastatic HCC cells exhibited considerably reduced viability in a dose-dependent manner following sorafenib treatment, regardless of whether it was used in combination with SERCA inhibitors (Fig. 4 A). Treatment with candidate 56 or 62 alone had a marginal anti-cancer effect on non-metastatic HCC cells (Fig. 4 A). Moreover, the viability of metastatic HCC cells was not significantly affected by the SERCA inhibitors or sorafenib treatment alone. In contrast, novel SERCA1-specific inhibitor candidate 56 and 62 used in combination with sorafenib remarkably reduced metastatic HCC cell viability in a dose-dependent manner (Fig. 4 B–D). SERCA is a pivotal player and therapeutic target in the regulation of cytosolic calcium overburden in cancer[ 15 , 34 ]. The anti-cancer effect of candidate 56 and 62 via the specific functional inhibition of SERCA1 was determined using microspectrofluorometry based on the differences in intracellular calcium levels in patient-derived non-metastatic and metastatic HCC cells treated with sorafenib alone or with SERCA inhibitors (Fig. 4 E–H). Free cytosolic calcium levels were measured in the presence of high-potassium depolarization and were estimated alone or combined sorafenib treatment with SERCA inhibitors (Thapsigargin; positive control, candidate 56, and candidate 62). Cytosolic free calcium levels in non-metastatic HCC cells failed to revert to basal levels following sorafenib treatment, regardless of whether it was used in combination with SERCA inhibitors (Fig. 4 E). Sorafenib treatment alone in metastatic HCC cells, the cytosolic free calcium levels revert to the basal levels. However, when SERCA1-specific inhibitors candidate 56 and 62 were combined with each, cytosolic free calcium failed to revert to basal levels (Fig. 4 F–H). There were no significant changes in the levels of cytosolic free calcium on treatment with sorafenib or SERCA inhibitors alone in metastatic HCC cells. These variations in cytosolic free calcium levels between patient-derived non-metastatic and metastatic HCC cells may be directly related to SERCA. To assess whether the prolonged survival of metastatic HCC cells on sorafenib treatment was indeed associated with only SERCA1 and not sodium-calcium exchangers (NCX), not calcium ion channels, not plasma membrane calcium ATPase (PMCA) we performed cell viability (Fig. 5 A–D) and immunoblot assays (Fig. 5 E) on treatment with NCX inhibitor (KB-R7943), calcium channel blockers (bepridil, verapamil or nifedipine), plasma membrane calcium PMCA inhibitor (caloxin2a1), and SERCA inhibitors in combination with sorafenib in non-metastatic and metastatic HCC cells (Fig. 5 A–E). In non-metastatic HCC cells, no significant changes were observed on treatment with either agent with csorafenib compared to that with sorafenib treatment alone (Fig. 5 A). In metastatic HCC cells, sorafenib treatment alone or combination treatment with NCX inhibitor, calcium channel blockers, PMCA inhibitor did not significantly influence survival, whereas combination treatment with SERCA inhibitors were failed to survive dose-dependently (Fig. 5 B, C, and D). Results of the immunoblot assay indicated that CHOP (an ER stress marker) levels increased significantly on combining SERCA inhibitors and sorafenib treatment in metastatic HCC cells (Fig. 5 E, top; right, bottom; left and right). However, in non-metastatic HCC cells, CHOP levels significantly increased in the presence of sorafenib regardless of treatment with an NCX inhibitor, calcium channel blocker, SERCA inhibitor, or PMCA inhibitor (Fig. 5 E, top; left). To verify the role of candidate 56 and 62 as SERCA1-specific inhibitors, we assessed the anti-cancer effects of candidate 56 or 62 when used in combination with each anti-cancer drug (sorafenib, 5-Fluorouracil [5-FU] and gemcitabine) in other metastatic HCC cells (YUMC-M-H8; patient-derived HCC cells were isolated from metastatic tissue of a patient after 5FU therapy, YUMC-M-H12; patient-derived HCC cells were isolated from metastatic tissue of a patient after gemcitabine therapy) (Fig. 5 F-I). mRNA quantification of metastatic HCC cells compare to non-metastatic HCC cells under each anti-cancer drug treated conditions showed increase in SERCA1 levels predominantly in YUMC-M-H1, -H2, and -H3; however, YUMC-M-H8 and YUMC-M-H12 showed significantly increased levels of the SERCA2 or SERCA3 isoforms respectively (Fig. 5 F). Protein quantification at the basal or anti-cancer drug (sorafenib, 5-FU and gemcitabine)-treated conditions using immunoblot assay further proved that the metastatic HCC cells, YUMC-M-H1, -H2, and -H3 significantly expressed SERCA1, whereas other metastatic HCC cells, YUMC-M-H8 and -H12 (patient-derived HCC cells, metastasis ocurred after 5-FU or gemcitabine therapy) significantly increased the SERCA2 and SERCA3 isoforms, respectively under anti-cancer drug treated conditions (Fig. 5 G). ER stress marker, CHOP levels were not significantly different in metastatic HCC cells regardless of the anti-cancer drug used. However, non-metastatic HCC cells showed significantly increased CHOP levels on sorafenib treatment (Fig. 5 G). In cell viability assays, combination treatment with the novel SERCA1 isoform-specific inhibitors (candidate 56 and 62) and anti-cancer drugs (sorafenib, 5-FU and gemcitabine), resulted in dominantly expressed SERCA1 (YUMC-M-H1, -H2, and -H3), SERCA2 (YUMC-M-H8), and SERCA3 (YUMC-M-H12), respectively, in the metastatic HCC cell line. Consequently, cell viability of SERCA1 dominantly expressed metastatic HCC cells was suppressed by SERCA1 isoform-specific inhibitors (candidate 56 and 62) with anti-cancer drug (Fig. 5 H, left), whereas SERCA2 or SERCA3 dominantly expressed HCC was no significantly influenced candidate 56 and 62 with anti-cancer drug (Fig. 5 H, right). Cell viability significantly decreased whereas CHOP levels increased in non-metastatic HCC cells on sorafenib treatment (Fig. 4 I). However, metastatic HCC cells (YUMC-M-H1, -H2, -H3, -H8, and -H12) did not significantly influence cell viability or ER stress under anti-cancer drug (sorafenib, 5-FU and gemcitabine) alone treated conditions. These cells showed significantly decreased cell viability and increased CHOP levels following treatment with the SERCA1 isoform-specific inhibitors, candidate 56 and 62 (Fig. 5 I). However, in SERCA2 and SERCA3 dominantly expressed HCC cells, YUMC-M-H8 and -H12, respectively, showed no significant difference in cell viability and increase in ER stress on combined treatment with anti-cancer drugs (5FU or gemcitabine) and candidate 56 or 62. Therefore, the novel SERCA inhibitors candidate 56 and 62 could be considered as SERCA1-specific inhibitors. These findings imply that SERCA1 regulates survival prolongation in metastatic HCC cells during chemotherapy treatment by mediating overloaded cytosolic free calcium levels through new SERCA1 isoform-specific inhibitors, candidate 56 and 62. A novel therapeutic approach for metastatic HCC through SERCA1-specific inhibitors (novel small molecules, candidate 56 and 62) in a patient-derived metastatic HCC cell mouse xenograft model We evaluated the anti-cancer effects of candidate 56 and 62 in vivo using a mouse xenograft tumor model with SERCA1, which predominantly increased patient-derived metastatic cells. We induced acute ER stress by treatment with sorafenib alone or with SERCA inhibitors. The dose of candidate 56 and 62 in xenograft model was selected in a dose-dependent manner following candidate 56 and 62 treatment with sorafenib (Supplementary Fig. 4A–C). In the xenograft model with non-metastatic HCC cells, conspicuous tumor shrinkage increased due to sorafenib treatment regardless of SERCA inhibitor combination treatment (Fig. 6 A, top). In the metastatic HCC xenograft model, sorafenib treatment alone did not increase tumor shrinkage; however, combinatorial treatment with sorafenib and candidate 56 or 62, novel SERCA1 isoform-specific inhibitors resulted in remarkable tumor shrinkage (Fig. 6 B–D, top). The excised tumors were similar in terms of tumor volume, which declined considerably with sorafenib treatment, regardless of the presence of SERCA inhibitors in the non-metastatic HCC cells (Fig. 6 A, middle). By comparison, dissected tumor weight in metastatic HCC cells was not meaningful influenced by each sorafenib alone, whereas combinatorial strategy to sorafenib with candidate 56 or 62 leaded a prominent decrease in tumor weight (Fig. 6 B–D, middle). All the inhibitory agents, administered alone or in combination, did not significantly influence the whole body weight of mice (Fig. 6 A–D, bottom). Further, morein the change of survival rate (Fig. 6 E) and whole body weight (Supplementary Fig. 4D) were measured under treatment with thapsigargin, candidate 56, or 62 alone in normal mice (not xenograft model) and compared with a non-treated group for 29 days. In normal mice, candidate 56 or 62 treatment alone showed no significant difference in whole body weight or death ratio compared with the group treated with thapsigargin alone (Fig. 6 E and Supplementary Fig. 4D). Tumor lysate-based immunoblot assay of non-metastatic HCC cells revealed that SERCA1 expression did not significantly change; however, CHOP expression was significantly induced with sorafenib treatment alone or in combination with SERCA inhibitors (Fig. 6 F). In contrast, metastatic HCC cells showed high SERCA1 expression following treatment with sorafenib alone or in combination with SERCA inhibitors (Fig. 6 G–I). Moreover, the sorafenib-induced overexpression of CHOP was significantly alleviated by an increase in SERCA1. However, the functional inhibition of SERCA1 using SERCA1-specific inhibitors, candidate 56 and 62 increased in acute ER stress (CHOP induction) (Fig. 6 G–I). Candidate 56 and 62, administered alone did not significantly influence the hepatic injury of mice compare than carbon tetrachloride alone treatment (CCl 4 , positive control) (Fig. 6 J). Moreover, candidate 56 and 62-mediated cardiac injury was no significantly induced in mice (Fig. 6 O–R). The increase in SERCA1 in metastatic HCC represents a targetable mechanism to overcome resistance. Consequently, in metastatic HCC, CaMK2α-mediated SERCA1 increase becomes a pivotal factor for survival under acute ER stress. These new small molecules, the SERCA1-specific inhibitors candidate 56 and 62, present a promising therapeutic approach for unmet medical needs without causing side effects and could potentially treat patients with anti-cancer drug-resistant-mediated metastatic and recurrent cancer at lower doses than those required for individual anti-cancer drug use. Discussion The advancement of anti-cancer drugs has led to numerous studies demonstrating the benefits of preoperative treatments in enhancing survival rates post-surgery[ 35 ]. Despite this progress, there remains a lack of established therapeutic options as standard neoadjuvant or adjuvant treatments for HCC[ 36 , 37 ], leading to a continual increase in unmet medical needs. A significant portion of these unmet needs is attributed to the recurrence and metastasis mediated by anti-cancer drug resistance[ 38 ]. Therefore, targeting anti-cancer drug resistant mediated-metastatic cancer cells remains a significant challenge in cancer treatment and research[ 39 , 40 ]. The mechanism of cancer metastasis in patients with cancer differs significantly for each cancer, owing to specific attributes that negatively affect cancer therapy and eventually cause cancer progression and death[ 40 , 41 ]. Consequently, a clinical solution for anti-cancer drug resistant-mediated metastatic cancer is an unattainable target. In the current study, we postulated a therapeutic framework for metastatic cancer based on the results of a prototypical model experiment. The ultimate goal of anti-cancer drugs is to induce cancer cell death. However, anti-cancer drug resistant-mediated metastatic cancer cells evade death through epigenetic reprogramming, which upregulates numerous target genes[ 42 ]. Among the various mechanisms of evading cell death, refractory cancer could be resistant to ER stress by evading excess cytosolic free calcium-mediated apoptosis under anti-cancer drug treatment or glucose starvation metabolic stress[ 9 ] and phenocopy cytotoxic chemotherapy-resistant cancers[ 8 , 16 , 23 ]. Conventional anti-cancer drugs target the increase in cytosolic free calcium, which causes calcium-mediated apoptosis in anti-cancer drug-sensitive cancer cells[ 43 ]. Management of excess cytosolic free calcium is critical in apoptosis-resistant cancer cells and is facilitated by the calcium pump[ 14 , 17 , 18 , 44 ]. In refractory cancers such as anti-cancer drug-resistant-mediated cancer cells many target genes are upregulated for survival under acute ER stress conditions[ 45 ]. SERCA is a critical target protein that supports low levels of resting cytosolic free calcium in cancer cells[ 46 ]. Moreover, enhanced SERCA expression is associated with poor outcomes in cancer, since it protects cells from excess cytosolic free calcium-mediated apoptosis[ 47 ]. The results of this study showed that, among the SERCA isoforms, SERCA1 levels predominantly increased in patient-derived metastatic HCC cells and further identified two novel small molecules, candidate 56 and 62, which were SERCA1-structure based specific inhibitors. mRNA sequencing (mRNA-Seq) analysis was based on the ECBS program, pharmacophore, and docking-based sequential virtual screening for identifying novel SERCA1-specific inhibitors, to devise a therapeutic strategy for refractory cancer. mRNA-Seq and immunoblot analyses revealed higher SERCA1 expression in metastatic HCC cells than that in non-metastatic HCC cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that Calcium and Notch signaling pathways ranked high among the top 10 signaling pathways in patient-derived metastatic HCC cells compared to those in patient-derived non-metastatic HCC cells. The correlation between Calcium and Notch signaling pathways is well-established[ 48 ], and Notch signaling is regulated by SERCA suppression[ 49 ]. Consequently, we identified SERCA isoforms and excess cytosolic free calcium-mediated apoptosis as key players in numerous upregulated target genes. The present study findings revealed that enhanced SERCA1 transcription through NFκB nuclear translocation by CaMK2α plays a pivotal role in evading excess cytosolic free calcium mediated apoptosis under acute ER stress conditions in anti-cancer drug (sorafenib)-induced cytotoxic stress-resistant HCC cells. Increase in SERCA1 levels by CaMK2α enhances the restoration of excess cytosolic free calcium, which mainly contributes to anti-cancer drug-resistance-mediated metastasis to anti-cancer drug treatment-elevated acute ER stress. Increase in SERCA1 expression by CaMK2α restrains excess calcium-dependent apoptosis. Notably, the current study proposes a reliable therapeutic approach for patient-derived metastatic HCC by identifying novel SERCA1-specific inhibitors, candidate 56 and 62. The present study findings validate the role of candidate 56 and 62, which could be prevent cardiac injury. SERCA isoforms are well-known key regulators of cardiac muscle, and therapeutic approaches to these SERCA inhibitors are unavoidably involved in cardiac dysfunction. Consequently, patients with SERCA-dependent anti-cancer drug-resistant-mediated metastatic cancer may also be concerned about cardiac dysfunction. However, identification and validation of SERCA isoform-specific inhibitors, as in the current study, may potentially decrease cardiac dysfunction. Findings of current study might be favorable to founding prospective, reasonable clinical approaches in patients with refractory HCC to advance efficacious theraphy. In a clinical relevance, these outcomes of current study impart significant implications for the development of novel combinatorial strategies and the discovery of new anti-cancer candidates that target a specific vulnerability of malignant cells, such as drug resistant-mediated etastatic cancer cells. However, several further studies were required to establish the current clinical approach. Furthermore, more study is needed owing to the limitations of several patient results. To overcome these limitations, several studies are on going on various types of patient-derived anti-cancer drug-resistant-mediated metastatic cancer. Abbreviations SERCA sarco/endoplasmic reticulum calcium ATPase CaMK2α Calcium/calmodulin-dependent protein kinase alpha NFκB nuclear factor kappa B RNA-Seq RNA sequencing qRT-PCR quantitative reverse transcription PCR CSCs cancer stem cells ER endoplasmic reticulum siRNA small interfering RNA RPMI-1640 Rosewell Park Memorial Institute-1640 FBS Fetal Bovine Serum YUMC Yonsei University Medical Center Declarations Acknowledgments We thank proffesor Chan Wung Kim (CKP Therapeutics, Inc., Massachusetts Medical Device Development Center, 110 Canal Street, Lowell MA 01852, USA) for supporting this research. Particulary we extend our sincere gratitude to Mung-Kun Park and Hyun Joo Hwang for their invaluable support in this research. Author Contributions JHL, KP, KHC, SMK, KCP and JHC primarily conducted the in vitro and in vivo studies and contributed to manuscript drafting. SMK and KCP isolated the patient-derived drug-resistant cancer cells. JMK, YLJ, KP and KHC performed statistical analyses. JHL, KP, KHC, SMK, KCP and JHC contributed to manuscript drafting and study design. JHL, SMK, KHC, KCP and JHC conceptualized the study, designed experiments, prepared the manuscript, and finalized it. Funding This study received support from a grant from the KHIDI, funded by the Ministry of Health & Welfare, Republic of Korea (HI18C1188), the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2017R1D1A1B03029716), and CKP Therapeutics, Inc. (grant numbers: 2021-31-1118), the Ministry of Trade, Industry & Energy (MOTIE), Korea Planning & Evaluation Institute of Industrial Technology (KEIT) through the Encouragement Program for Technology Development (Project NO: RS-2024-00410585). Data availability No datasets were generated or analysed during the current study. Ethics approval and consent to participate The research protocol was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (IRB Protocol: 3-2022-0331). Cell samples were obtained from patients at the Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea. This study was performed in accordance with the Declaration of Helsinki. The requirement for obtaining informed consent was waived owing to the retrospective nature of the study. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files, or available from the corresponding author upon reasonable request. Competing Interests The authors declare that they have no competing interests. References Alqahtani A, Khan Z, Alloghbi A, Said Ahmed TS, Ashraf M, Hammouda DM. 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Pagliaro L, Marchesini M, Roti G. Targeting oncogenic Notch signaling with SERCA inhibitors. J Hematol Oncol. 2021;14(1):8. Tables Table 1 Properties and clinical features of patients. Patient-derived Hepatocellular carcinoma cells were isolated from these patient specimen. YUMC-NM-H1 YUMC-M-H1 YUMC-M-H2 YUMC-M-H3 Age at Diagnosis 71 53 67 71 Gender Female Female Male Male Primary Disease Site Liver Liver Liver Liver Stage T1bN0M0 T3N0M1 T1N1M1 T2N1M1 Primary Pathology Hepatocellular carcinoma Hepatocellular carcinoma (Metastasis after sorafenib treatment) Hepatocellular carcinoma (Metastasis after sorafenib treatment) Hepatocellular carcinoma (Metastasis after sorafenib treatment) Classification of specimen used for culture Fresh tumor Fresh tumor Fresh tumor Fresh tumor Obtained from Severance Hospital, Seoul, Korea Severance Hospital, Seoul, Korea Severance Hospital, Seoul, Korea Severance Hospital, Seoul, Korea Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7382767","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515779708,"identity":"b2cf593e-d2a0-4ef3-ad56-3e7b960dbfc0","order_by":0,"name":"Jin Hong Lim","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"Hong","lastName":"Lim","suffix":""},{"id":515779709,"identity":"ecebebe0-f0d3-447d-ac6f-2de2ed5c5e00","order_by":1,"name":"Keunwan Park","email":"","orcid":"","institution":"KIST Gangneung Institute of Natural Products","correspondingAuthor":false,"prefix":"","firstName":"Keunwan","middleName":"","lastName":"Park","suffix":""},{"id":515779710,"identity":"d76eadb4-099e-476e-bdea-ba245ad29101","order_by":2,"name":"Kyung Hwa Choi","email":"","orcid":"","institution":"CHA Bundang Medical Center, CHA University","correspondingAuthor":false,"prefix":"","firstName":"Kyung","middleName":"Hwa","lastName":"Choi","suffix":""},{"id":515779711,"identity":"c16b65dc-297b-48f9-a900-92db5e2b1fd9","order_by":3,"name":"JungMin Kim","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"JungMin","middleName":"","lastName":"Kim","suffix":""},{"id":515779712,"identity":"ef806931-499b-43f7-815f-2d97528ebfa4","order_by":4,"name":"Yoo-Lim Jhe","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoo-Lim","middleName":"","lastName":"Jhe","suffix":""},{"id":515779713,"identity":"5477f507-90a1-4678-8ca4-b987a95b6b8e","order_by":5,"name":"Seok-Mo Kim","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Seok-Mo","middleName":"","lastName":"Kim","suffix":""},{"id":515779714,"identity":"2855d3bf-ab2d-4986-b77a-f58f3132bf7d","order_by":6,"name":"Ki-Cheong Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYFACxgYGhop/cgwMPCRpOXPAmBQtIF1tBxIbiNYi77+47TNv2530DTdyD39gqLEjrMXwxsPm2TznnuVuuJGXJsFwLJkILTMONjPzlDEDteSYMTCwHSBWCxtzusGNHOMPDP+I0CLP3wjU0nY4AajFQAIYDoS1AJU1M845k2Y488wbM4nEPiL8It9//DHDmwobeb7jQId9+EZEiBncSIAwFEBOSiCsAWTLASijgRjlo2AUjIJRMCIBAEGnPwfJsC1YAAAAAElFTkSuQmCC","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Ki-Cheong","middleName":"","lastName":"Park","suffix":""},{"id":515779715,"identity":"2bb89ea3-ecf6-4f82-975e-5d1f2ff3f061","order_by":7,"name":"Jae-Ho Cheong","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jae-Ho","middleName":"","lastName":"Cheong","suffix":""}],"badges":[],"createdAt":"2025-08-15 16:08:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7382767/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7382767/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91524898,"identity":"ac419d42-0767-497f-b4f6-9f273cdd2ff3","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":789211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignaling pathway and target gene expression profiling in patient-derived HCC cells under basal or sorafenib-mediated endoplasmic reticulum (ER) stress conditions. A,\u003c/strong\u003e Hierarchical clustering shows differential gene expression; gene expression profiles in the metastatic HCC cells compared to those in patient-derived non-metastatic HCC cells. B, Bar plot revealed 10 prominently stimulated pathways in the patient-derived metastatic HCC cells compared to those in patient-derived non-metastatic HCC cells. \u003cstrong\u003eC,\u003c/strong\u003e Immunoblot analysis for validation of HCC or metastatic marker expressions between the non-metastatic and metastatic HCC cells compare than normal liver cell. D, Alterations of the target genes through immunoblot assay under basal or sorafenib-mediated sever ER stress condition between non-metastatic and metastatic HCC cells. \u003cstrong\u003eE–H,\u003c/strong\u003e Gene expression variances were evaluated on mRNA sequencing (mRNA seq) analysis between the non-metastatic and metastatic HCC cells. E, Each assay was performed at least in triplicate. *p\u0026lt;0.05 and **p\u0026lt;0.01 versus non-metastatic HCC, YUMC-NM-H1.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/16a769ea40c6d443abbe6a6f.png"},{"id":91524897,"identity":"e02de699-bce6-4663-8a49-02b229f9dd6e","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1285504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePatient-derived metastatic HCC cell survival was prolonged through the nuclear translocation of nuclear factor kappa B (NFκB) by calcium/calmodulin-dependent protein kinase alpha (CaMK2α), which mediated an increase in SERCA1 levels under sorafenib-treated conditions. A,\u003c/strong\u003e Immunoblot analysis revealed that the regulation of nuclear translocation of nuclear factor kappa B (NFκB) by CaMK2α influenced proteins related to calcium restoration (SERCA1), ER stress (CHOP), apoptosis (cleaved caspase 3), and anti-apoptosis (Bcl-2) under the presence or absence of sorafenib between non-metastatic and metastatic HCC cells. \u003cstrong\u003eB and C\u003c/strong\u003e, Immunoblot analysis (B) and cell viability assay (C) of CaMK2α-silenced metastatic HCC cells, YUMC-M-H1, -H2, and -H3 on treatment with sorafenib (left; YUMC-M-H1, middle; YUMC-M-H2, right; YUMC-M-H3). *p\u0026lt;0.05 and **p\u0026lt;0.01 versus control (unmodified metastatic HCC cells, YUMC-M-H1, -H2, and -H3 under sorafenib treatment). \u003cstrong\u003eD\u003c/strong\u003e, Immunofluorescence assay for nuclear expression of NFκB in CaMK2α-silenced metastatic HCC cells under sorafenib-treated conditions. Magnification, × 400. Scale bar, 20 μm. Data represent the average of at least three separate independent experiments and are presented as the means ± SEM. \u003cstrong\u003eE,\u003c/strong\u003e Cytosolic free calcium content in CaMK2α-silenced metastatic HCC cells, YUMC-M-H1, -H2, and -H3 under sorafeni treatment. The red arrow shows the high potassium depolarization which induces an increase in cytosolic calcium. Each assay was performed at least in triplicate.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/1db2e57b8c30a30a970bc625.png"},{"id":91524901,"identity":"09d116fe-2231-4f60-9da1-8c28662c6916","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1449426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and molecular docking models of candidate 56 and 62 for SERCA1.\u003c/strong\u003e \u003cstrong\u003eA, \u003c/strong\u003eScreening models for identification of candidate 56 and 62 in patient-derived metastatic HCC cells. \u003cstrong\u003eB,\u003c/strong\u003e The candidate 56 and 62 carbons are represented by cyan and yellow, respectively. \u003cstrong\u003eC,\u003c/strong\u003e The docking sites for candidate 56 and 62 in the cytosolic N domain of SERCA1. \u003cstrong\u003eD,\u003c/strong\u003e The N, P, and A domains are annotated and the binding residues are shown in blue. \u003cstrong\u003eE \u003c/strong\u003eand\u003cstrong\u003e F,\u003c/strong\u003e candidate 56 or 62 binding residues in SERCA1 are represented as sticks in green. The transmembrane regions are represented by a grey ribbon. The enlarged representation of protein-ligand interactions in the docking models highlight the binding residues involved in interactions with candidate 56 and 62. The pi-pi stacking of the compounds with F487 is indicated by a dotted yellow line. The different colors used in the Fig. correspond to the properties of the binding site residues. \u003cstrong\u003eG,\u003c/strong\u003eThe alignment of (predicted) human SERCA1 and (X-ray crystal) rabbit SERCA1 structures are represented by cartoons (root mean square deviation of 1.89 Å). The human SERCA1 structure is shown in white, whereas the rabbit SERCA1 structure is shown in blue. The ATP-derivative bound to the rabbit SERCA1 structure (PDB ID 6YSO) is illustrated with red sti4Hcks, alongside the docking conformations of candidate 56 and 62 shown in cyan and yellow, respectively. \u003cstrong\u003eH\u003c/strong\u003e, Pharmacophore models based on the receptor–ligand interactions.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/09b8e95509aac1d4336d26f2.png"},{"id":91524899,"identity":"b5e2f954-e9dd-46e9-af8d-9ba8dc2f7a93","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":629070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability and cytosolic free calcium measurement in whole patient-derived HCC cells after thapsigargin (SERCA inhibitor, positive control) and molecular docking models of candidate 56 and 62 for SERCA1. A,\u003c/strong\u003e The candidate 56 and 62 carbons are represented by cyan and yellow, respectively. \u003cstrong\u003eB,\u003c/strong\u003e The docking sites for candidate 56 and 62 in the cytosolic N domain of SERCA1. \u003cstrong\u003eC–F,\u003c/strong\u003e Cell viability assay between non-metastatic and -metastatic HCC cells on treatment with sorafenib and SERCA inhibitors alone or combination, excluding combination with SERCA inhibitors. \u003cstrong\u003eG–J,\u003c/strong\u003e Cytosolic free calcium measurement between non-metastatic and metastatic HCC cells on treatment with sorafenib and SERCA inhibitors alone or combination, excluding combination with SERCA inhibitors. The red arrow shows high potassium depolarization addition to increase cytosolic calcium. \u003cstrong\u003eG,\u003c/strong\u003e non-metastatic HCC cells, YUMC-NM-H1. \u003cstrong\u003eH–J,\u003c/strong\u003e metastatic HCC cells, YUMC-M-H1, -H7, and -H8. *p\u0026lt;0.05 and **p\u0026lt;0.01 versus control, *,**; thapsigargin, *,**; candidate 56, *,**; candidate 62. T; thapsigargin, S; sorafenib, C56; candiate 56, C62; candidate 62. Each assay was performed at least in triplicate.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/00e0904b4a4a3e36f7ea1c2c.png"},{"id":91524902,"identity":"1928f3f8-6046-457b-a42c-62718a7e51ac","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":923736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability assay in whole patient-derived HCC cells after treatment with SERCA1 isoform specific novel candidate (candidate 56 and 62) treatment in the presence of anti-cancer drug (sorafenib, 5FU and gemcitabine).\u003c/strong\u003e \u003cstrong\u003eA–D,\u003c/strong\u003e Dose-dependent cell viability in the presence of calcium channel blocker (bepridil, verapamil, or nifedipine), sodium-calcium exchangers (NCX) inhibitor (KB-R7943), plasma membrane calcium ATPase (PMCA) inhibitor (caloxin 2a1), and SERCA inhibitors (thapsigargin, candidate 56 and 62) with sorafenib between non-metastatic and -metastatic HCC cells. *p\u0026lt;0.05 and **p\u0026lt;0.01 versus control (the absence of sorafenib), **; thapsigargin, *,**; candidate 56, *,**; candidate 62, S; sorafenib. \u003cstrong\u003eE,\u003c/strong\u003eImmunoblot analysis for ER stress marker induction under combination treatment with the various inhibitors and sorafenib between non-metastatic and metastatic HCC cells. \u003cstrong\u003eF,\u003c/strong\u003e Differential SERCA isoform-dependent RNA expression in the metastatic (YUMC-M-H1, -H2, and -H3), 5FU-resistant-mediated metastatic HCC (YUMC-M-H8), and gemcitabine-resistant mediated (YUMC-M-H12) cells compared to those in non-metastatic HCC cells. S; sorafenib, G; gemcitabine. *p\u0026lt;0.05 and ** p\u0026lt;0.01 versus non-metastatic HCC cells. \u003cstrong\u003eG,\u003c/strong\u003e Immunoblot analysis for the differential dominance of SERCA isoforms-mediated induction of ER stress marker between non-metastatic and metastatic (sorafenib, 5FU or gemcitabine-resistant mediated HCC) cells under each agent presence or absence conditions. \u003cstrong\u003eH and I,\u003c/strong\u003e Cell viability (H) and immunoblot assay (I) for the selective suppression of viability or selectively inducing ER stress under combination treatment of candidate 56 or 62 with sorafenib, 5FU or gemcitabine in non-metastatic and metastatic (sorafenib, 5FU or gemcitabine-resistant mediated HCC) HCC cells. S; sorafenib, C56; candidate 56, C62; candidate 62, G; gemcitabine. *p\u0026lt;0.05 versus control (sorafenib only treatment). Each assay was performed at least in triplicate.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/a58ee2009ba4a249237ca85e.png"},{"id":91524900,"identity":"c7c8b2b8-7fbb-4179-8824-86c8cdda37cc","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1921259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCandidate 56 and 62 suppressed tumor growth in a xenograft model of patient-derived metastatic HCC cells.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e, non-metastatic HCC cells (YUMC-NM-H1),\u003cstrong\u003eB, C and D \u003c/strong\u003emetastatic (YUMC-M-H1, -H2, and -H3) HCC cells, Changes in relative tumor volumes (top), resected tumor weights (middle), and whole body weights (bottom) of patient-derived HCC cells (each group, n = 10).\u003cstrong\u003e \u003c/strong\u003eTumor sizes were calculated in NOD/Shi-scid, IL-2Rγ KOJic (NOG) mice. Animals were treated with each agent alone or sorafenib combined with the SERCA inhibitors, thapsigargin, candidate 56, and 62. Data represent the mean ± standard error of the mean. *.# p\u0026lt; 0.05 and **,## p\u0026lt; 0.01, compared with the control. #, ##; versus control, *,**; thapsigargin control, *,**; candidate 56, *,**; candidate 62, T; thapsigargin. \u003cstrong\u003eE,\u003c/strong\u003e The change of death ratio measurement under treatment with thapsigargin, candidate 56 or 62 alone in normal mice (not xenograft model) compared with the non-treated group (control). \u003cstrong\u003eF–I,\u003c/strong\u003eImmunoblot assay for the induction of SERCA isoforms (1–3) and ER stress under combination treatment SERCA1-specific inhibitors, candidate 56 and 62 with sorafenib in a mouse xenograft model with patient-derived non-metastatic and metastatic HCC cells. \u003cstrong\u003eI–M and O–R, \u003c/strong\u003eThe histological change of the liver and heart similar treatment as shown by Masson's trichrome staining. Nuclei are stained black, cytoplasm and muscle fibres red, and ECM components blue.\u003cstrong\u003e \u003c/strong\u003eAll slides were examined at 200 × magnification; scale bar: 100 μm. Each assay was performed in triplicate, and the representative images are presented. C56; candiate 56, C62; candidate 62. Each assay was performed at least in triplicate.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/4bf054ca8d40011d62e72b77.png"},{"id":91527525,"identity":"0058985a-4276-40fc-801a-a8328d2ab45b","added_by":"auto","created_at":"2025-09-17 11:19:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8740725,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/8436c538-8b67-4b01-b645-e318936b4fbb.pdf"},{"id":91526665,"identity":"769ba884-48aa-42e8-9532-db7c10b45d0c","added_by":"auto","created_at":"2025-09-17 11:11:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3580868,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/ec3dba2049d0fb758a7b3ac8.docx"},{"id":91524896,"identity":"ce5ce9c7-28c5-4e5d-909d-09bb8b7e1dbd","added_by":"auto","created_at":"2025-09-17 11:03:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16423,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7382767/v1/478570c2ebc291f88c22481c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discveory of SERCA1 specific small molecule inhibotirs based on the survival mechanisms of metastatic hepatocellular carcinoma cells dependent on CaMK2α-Mediated SERCA1 expression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is a well-known form of liver cancer, comprising over 90% of hepatic carcinoma cases. Fortunately, advancements in systemic treatments for advanced HCC have led to a steady increase in patient survival rates[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the emergence of anti-cancer drug resistance in some cases presents a critical challenge, leading to patient mortality due to cancer recurrence and metastasis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Molecular variations suggest the poor prognosis of HCC in patients with metastasis or recurrence; however, the fundamental mechanisms remain unclear. Therefore, more efficient and reliable therapeutic approaches are required. Recently, the ratio of relapse to survival after surgery was determined following systemic chemotherapy before surgery, regardless of cancer development[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Systemic chemotherapy is typically administered to patients with advanced cancer subtypes characterized by invasion or metastatic lesions, as local therapies are often inadequate against chemotherapy-resistant cancer[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Anti-cancer drug treatments exert significant stress on cancer cells, contributing to cellular viability challenges. Cancer stem cells (CSCs) have demonstrated resilience against metabolic stresses within tumor microenvironments through epigenetic reprogramming, emerging as a notable therapeutic target[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Under anti-cancer drug treatment conditions, CSCs exhibit enhanced survival mechanisms against acute sarco/endoplasmic reticulum (ER) stress compared to non-CSCs, prompting the exploration of various molecular strategies for epigenetic reprogramming[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Epigenetic reprogramming stimulated by anti-cancer drugs may involve several molecular regulators[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Previous studies have established calcium/calmodulin-dependent protein kinase alpha (CaMK2α) as a pivotal transcriptional regulator that inhibits cytosolic free calcium-mediated apoptosis by upregulating sarco/ER calcium ATPase (SERCA) levels[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, CaMK was a key transcriptional regulator of increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression, which is a potent inducer of ATP production via mitochondrial respiration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cytosolic free calcium levels play a critical role in cellular responses to ER stress and subsequent cell fate decisions[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ER stress induces the release of cytosolic free calcium from the ER to the cytosol via IP3R (inositol 1,4,5-trisphosphate) receptors, which is regulated by calcium pumps, exchangers, and channels to maintain cellular calcium homeostasis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Excessive elevation of cytosolic free calcium beyond physiological levels triggers apoptotic signals[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. SERCA serves as a key regulator in managing overloaded cytosolic free calcium levels in cancer cells, contributing to cellular defense by efficiently restoring cytosolic free calcium to the ER[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previously, we showed potential implications for applying new combinatorial strategies and discovering anti-cancer candidates that SERCA-targeted a specific vulnerability of anti-cancer drug-resistant-mediated cells[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, a cardiac dysfunction was inevitable in xenograft model because of non-specific inhibition of SERCA isoforms by several candidates[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Injured myocardial calcium cycling is a key regulator of heart failure (cardiac dysfunction), causing to a change in the structure remodelling and contractile function of the heart[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In cardiomyocytes, the regulation of cytosolic free calcium storage and release by sarcoplasmic reticulum (SR) is mostly dependent on calcium regulation proteins, such as SERCA2a. For the relaxation phase of the cardiac cycle, SERCA2a is a pivotal regulator in transporting cytosolic free calcium back to the SR, to restore cytosolic free calcium levels to their resting state and replenish SR calcium levels for the next contraction. However, functional inhibition of SERCA2a by thapsigargin (non SERCA isoform specific inhibitor) causes to cardiac dysfunction[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The current study aimed to design a therapeutic approach with decrease of cardiac dysfunction based on CaMK2α-mediated enhancement of SERCA1 via the regulation of cytosolic free calcium levels. We further aimed to identify targets and novel small molecules for isoform-specific inhibition of metastatic HCC under acute ER stress in patient-derived samples. The findings of this study are clinically significant for the development of new small molecules with decrease of side effects that selectively and effectively target highly malignant cancer cells, including cancer stem-like cells and drug-resistant cancer cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient characteristics\u003c/h2\u003e\u003cp\u003eThe patients recruited in this study underwent liver resection. They were treated between January 2018 and December 2022 at the Severance Hospital (Seoul, South Korea). Details of patient presentations, surgical and pathological findings, and outcomes were obtained from the Cancer Center Database. All procedures involving patients were performed in accordance with institutional ethical standards, applicable local/national regulations, and guidelines of the 1964 Helsinki Declaration and its later amendments. Further details are provided in Supplementary Methods.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePatient tissue specimens\u003c/h3\u003e\n\u003cp\u003eTumor specimens were obtained from patients with biochemically and histologically confirmed recurrent or metastatic tumors who were treated at Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Fresh tumor specimens were obtained for surgical resection.\u003c/p\u003e\n\u003ch3\u003eEthical considerations\u003c/h3\u003e\n\u003cp\u003e The study protocol was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (Institutional Review Board Protocol: 3-2022-0331). Samples were obtained from patients at Severance Hospital, Yonsei University College of Medicine, Seoul, South Korea. The requirement for obtaining formal written consent was waived owing to the retrospective observational nature of the study. Further patient details are provided in the Supplementary Methods.\u003c/p\u003e\n\u003ch3\u003eTumor cell isolation and primary culture\u003c/h3\u003e\n\u003cp\u003eAfter resection, the tumors were kept in normal saline containing antifungal agents and antibiotics and moved to the laboratory. Normal tissues and fat were removed, and the tissues were rinsed with 1\u0026times; Hank\u0026rsquo;s Balanced Salt Solution. The cells were resuspended in Rosewell Park Memorial Institute (RPMI)-1640 medium (Hyclone, South Logan, UT, USA) with 20% FBS (Hyclone) and 2% penicillin/streptomycin solution (Gibco, Grand Island, NY, USA). Further details of the protocol have been Supplementary Methods or previously published[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe patient-derived cancer cells, THLE-2, Huh-7, HepG2 and Hep3B cells were obtained from fresh tumors of patients or ATCC (ATCC, Manassas, VA, USA). YUMC-NM-H1 (non-metastatic HCC) and YUMC-M-H1, -H2, and -H3 (metastatic HCC) cells were isolated from patients with HCC treated at the Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Cells were grown in RPMI-1640 medium containing 20% FBS. Cells were authenticated using short tandem repeat profiling, karyotyping, and isoenzyme analysis. Mycoplasma contamination was evaluated using a Lookout Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, MO, USA; MP0035).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003emRNA sequencing and analysis\u003c/h2\u003e\u003cp\u003eWhole RNA was isolated from the patient-derived cancer cells, YUMC-NM-H1 (non-metastatic HCC) and YUMC-M-H1, -H2, and \u0026ndash;H3 (metastatic HCC), using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions. Paired-end sequencing reads were generated on the Illumina sequencing NovaSeqX platform. Before starting analysis, Trimmomatic v0.38 was used to remove adapter sequences and trim bases with poor base quality. RNA enrichment (total RNA) in current study, we preprocessed the raw reads from the sequencer to remove low quality and adapter sequences before analysis and aligned the processed reads to the \u003cem\u003eHomo sapiens\u003c/em\u003e genome assembly (GRCh38) using HISAT v2.1.0 (HISAT2, RRID: SCR 015530)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. HISAT utilizes two types of indexes for alignment: a global, whole-genome index, and tens of thousands of small local indexes. Both are constructed using the same Burrows\u0026ndash;Wheeler transform (BWT) or graph FM index (GFM) as Bowtie2 (Bowtie 2, RRID: SCR 016368). The reference genome sequence of Homo sapiens (GRCh38) and annotation data were downloaded from the National Center for Biotechnology Information (NCBI). Aligned data (SAM file format) were sorted and indexed using SAMtools v 1.9. After alignment, transcript assembly of known transcripts was processed using StringTie v2.1.3b (StringTie, RRID: SCR016323)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Based on these results, expression abundance of transcript and gene were calculated as read count or fragments per kilobase of exon per million fragments mapped (FPKM) value per sample.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStatistical analysis of gene expression level\u003c/h3\u003e\n\u003cp\u003eThe relative abundances of genes were measured in Read Count using StringTie. We performed statistical analyses to find differentially expressed genes using the estimates of abundances for each gene in the samples. Genes with more than one Read Count values in the samples were excluded. To facilitate log2 transformation, 1 was added to each Read Count value of filtered genes. Filtered data were log2-transformed and subjected to trimmed mean of M-values (TMM) normalization. The statistical significance of the differential expression data was determined using exactTest, edgeR and fold change, in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting the p-value using the Benjamini-Hochberg algorithm. However statistical analysis in current study was used raw p-value instead to FDR. Because if used FDR, not acquired sufficiently gene list for analysis of enrichment for GO and KEGG. For differentially expressed genes (DEGs) sets, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Gene-enrichment and functional annotation analysis and pathway analysis for significant gene list were performed based on Gene Ontology and KEGG pathway analyses.\u003c/p\u003e\n\u003ch3\u003esiRNA transfection for CaMK2α knockdown\u003c/h3\u003e\n\u003cp\u003ePatient-derived metastatic HCC cells, YUMC-M-H1, -H2, and -H3 cells were transfected with CaMK2α siRNA or scrambled control siRNA. Sequence of the CaMK2α siRNA was designed using si-Designed software (Bioneer, Daejeon, South Korea) and the siRNA duplex was purchased from Bioneer. The sense and antisense sequences of the siRNA were as follows: 5'-UGAUCGAA GCCAUAAGCAA(dTdT)-3' (forward) and 5'-UUGCUUAUGGCUUCGAUCA(dTdT)-3' (reverse). Further details are provided in our previously published article[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSubcellular fractionation and immunoblot analysis\u003c/h2\u003e\u003cp\u003ePrimary antibodies against GPC3 (1:500, Cat#174851, Abcam, Cambridge, UK), HSP70 (1:500, Cat#2787, Abcam), TP (1:500, Cat#226917, Abcam), SERCA1 (1:200, Cat#133275, Abcam), SERCA2 (1:400, Cat#137020, Abcam), SERCA3 (1:200, Cat#154259, Abcam), Bcl-2 (1:400, Cat#196495, Abcam), pCaMK2α (1:500, Cat#171095, Abcam), CaMK2α (1:500, Cat#92332, Abcam), pIKKα (1:500, Cat#138426, Abcam), CHOP (1:500, Cat#7351, Santa Cruz Biotechnology, CA, USA), pNFκB (1:500, Cat#86299, Abcam), Histone H3 (1:500, Cat#18521, Abcam), caspase-3 (1:500, Cat#9661, Cell Signaling Technology, Beverly, MA,USA) and β-actin (1:500, Cat#47778) were used. Equal amounts of protein were separated on an 8\u0026ndash;10% sodium dodecyl sulfate-polyacrylamide gel, and the resolved proteins were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were subsequently blocked with 5\u0026ndash;10% nonfat milk in TBST for 1 h at room temperature or overnight at 4\u0026deg;C and incubated with appropriate concentrations of primary antibodies overnight at 4\u0026deg;C. The membranes were then rinsed 3\u0026ndash;10 times with TBST and probed with the corresponding secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) at room temperature for 1 h. After rinsing, the blots were developed with ECL reagents (Pierce) and exposed to Kodak X-OMAT AR Film (Eastman Kodak, Rochester, NY, USA) for 1\u0026ndash;5 min. Nuclear fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scientific; #78833) in accordance with the manufacturer's instructions. Separated nuclear and cytoplasmic extracts were isolated using a protein extraction solution (PRO-PREP, iNtRON Biotechnology, Seoul, Korea, #17081) or a histone extraction kit (Abcam, Cambridge, UK, #113476). Protein extracts and bands were quantified using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and the ImageJ software (NIH, Bethesda, MD, USA). Further details are provided in the Supplementary Methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eElectrophoretic mobility shift assay (EMSA)\u003c/h2\u003e\u003cp\u003eThe DNA binding activity of NFκB against the SERCA1 promoters was confirmed using a \u003csup\u003e32\u003c/sup\u003eP-labelled oligonucleotide. Specific labelled and unlabelled oligonucleotides are as follows. The DNA binding activity of NFκB against the \u003cem\u003eSERCA1\u003c/em\u003e promoter was confirmed using a \u003csup\u003e32\u003c/sup\u003eP-labelled oligonucleotide. Labelled and unlabelled oligonucleotides specific for NFκB (Forward 5\u0026prime;-GGGGGGTTCCC-3\u0026prime;, Reverse 5\u0026prime;-GGGAACCCCCC-3\u0026prime;) and mutant NFκB (Forward 5\u0026prime;-GGGcGcTTCCC-3\u0026prime;, Reverse 5\u0026prime;-GGGAAgCgCCC-3\u0026prime;) were synthesized by Bioneer. Further protocol details are as described in Supplementary Methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDual Luciferase Reporter Assay\u003c/h2\u003e\u003cp\u003ePromoter activity was evaluated using the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA; E1960), according to the manufacturer's protocol. Regions of NFκB binding sites were amplified by PCR from human genomic DNA (NFκB primers: Forward 5\u0026prime;-GGGGGGTTCCC-3\u0026prime;, Reverse 5\u0026prime;-GGGAACCCCCC-3\u0026prime;). The PCR products were cloned into the pGL4.70 promoter Vector (Promega) using T4 DNA ligase (Thermo Scientific, Waltham, MA, USA; EL0011). All insertions were confirmed by sequencing. Further protocol details are as described in Supplementary Methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence analysis and confocal imaging\u003c/h2\u003e\u003cp\u003eThe expression level of nuclear translocated pNFκB was analyzed by immunofluorescence staining. Cells cultured on glass-bottomed dishes (MatTek, Ashland, MA. USA) were fixed with 4% formaldehyde solution (R\u0026amp;D Systems, Abingdon, UK) for 10 min and permeabilized with 0.5% TritonX-100 in phosphate-buffered saline (PBS) for 10 min. The slides were air dried, washed with PBS, and incubated overnight at 4\u0026deg;C with anti-pNFκB (1:50, Cat#86299, Abcam, Cambridge, UK) in 3% bovine serum albumin included PBS. Further details are provided in the Supplementary Methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCytosolic free calcium measurements using microspectrofluorimetry\u003c/h2\u003e\u003cp\u003eCytosolic free calcium levels in patient-derived HCC cells were imaged using a calcium-sensitive fluorescent dye, Fura-2AM. HCC cells were incubated with Fura-2AM in normal phosphate buffered saline solution for 30 min at 37 ℃, followed by de-esterification of the dye for another 30 min at 22\u0026ndash;25 ℃. Further details are provided in a previously published article[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCell viability assay\u003c/h2\u003e\u003cp\u003eCell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were seeded in 96-well plates at a density of 6 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and cultured overnight to achieve 80% confluency. Further details on the protocol are available in existing publications[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The assay was performed thrice and cell viability was evaluated as a percentage of the signal observed in vehicle-treated cells and reported as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking simulation for SERCA1\u003c/h2\u003e\u003cp\u003eThe three-dimensional structure of SERCA1 was prepared using AlphaFoldDB[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] with the UniProt ID O14983. Most of the residues in the alpha-fold-predicted structure had high confidence scores, confirming reliability of the prediction model. The modeled human SERCA1 structure was further refined using Rosetta Relax[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] for use in subsequent docking simulations to elucidate crucial ligand-receptor interactions. The docking simulations for the two active compounds were conducted using DiffDock, which exhibits superior performance in blind docking tasks (i.e., docking simulation without knowing prior binding site information) The docked complex model was refined using RosettaLigand by performing iterative local docking refinement and energy minimization[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The final docking model was selected based on the interface binding energy (interface delta score) and used to analyze detailed molecular interactions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eTotal RNA extraction and quantitative reverse transcription-polymerase chain reaction\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from tumor cells using the RNeasy Mini Kit (Qiagen, cat. 74106) and One-Step reverse transcription-polymerase chain reaction (RT-PCR) Kit (Qiagen, Cat#204243) according to the manufacturer\u0026rsquo;s protocols. All data were normalized to \u003cem\u003eα-tubulin\u003c/em\u003e expression. The following primers for \u003cem\u003eSERCA1, SERCA2\u003c/em\u003e, and \u003cem\u003eSERCA3\u003c/em\u003e were used for quantitative RT-PCR (qRT-PCR) analysis: \u003cem\u003eSERCA1\u003c/em\u003e, 5'-GTGATCCGCCAGCTAATG-3' (forward) and 5'-CGAATGTCAGGTCCGTCT-3' (reverse); \u003cem\u003eSERCA2\u003c/em\u003e, 5'-GGTGGTTCATTGCTGCTGAC-3' (forward) and 5'-TTTCGGACAAGCTGTT GAGG-3' (reverse); \u003cem\u003eSERCA3\u003c/em\u003e, 5'-GATGGAGTGAACGACGCA-3' (forward) and 5'-CCA GGTATCGGAAGAAGAG-3' (reverse); and α-tubulin; 5'- CGGGCAGTGTTTGTAGACTTGG-3' (forward) and 5'-CTCCTTGCCAATGGTGTAGTGC-3' (reverse).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003emouse xenograft model with patient-derived HCC cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All the experiments were performed in accordance with the guidelines established by the Animal Experimentation Committee of Yonsei University. Patient-derived HCC cells (4.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mouse) were injected subcutaneously into the upper left flank region of female NOD/Shi-scid, IL-2Rγ KOJic (NOG) mice. Animals were maintained under specific pathogen-free conditions. All experiments were approved by the Animal Experiment Committee of Yonsei University (IACUC approval No 2022\u0026thinsp;\u0026minus;\u0026thinsp;0105). The detailed protocol is described in the Supplementary Methods.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMasson trichrome and H\u0026amp;E (Hematoxylin and Eosin) staining for establishment to hepatic and cardiac injury\u003c/h2\u003e\u003cp\u003eHepatic and cardiac biopsy specimens fixed in 10% buffered formalin solution were embedded with paraffin. Sections (5 \u0026micro;m) were stained with masson trichrome or H\u0026amp;E. The degree of cellular changes in hepatic and cardiac injury was evaluated with masson trichrome or H\u0026amp;E staining according to the protocol provided by the manufacturer protocol. The procedure stains nuclei dark red, cytoplasm and muscle fibres red, and ECM components blue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 6.0; GraphPad Software, La Jolla, CA, USA), and immunohistochemistry results were evaluated using analysis of variance, followed by the Bonferroni post-hoc test. Values are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ePatients and disease characteristics\u003c/h2\u003e\u003cp\u003eThis study targeted and isolated cancer cell from speciment of cancer patients who took anti-cancer drugs but caused metastasis. The patient group consists of nine men and five women, a total of fourteen (Supplementary Fig.\u0026nbsp;1A and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to patient-derived non-metastatic (included Huh7) or metastatic HCC cells, metastatic HCC cells showed increase of metastatic markers (\u003cem\u003eNDUFA4L2, EBF1, TYMP [Thymidine phosphorylase] and MET\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;1B), cancer stemness markers (\u003cem\u003ePROM1 [CD133], ALDH1A1, KRT17 and KRT19\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;1C) and EMT markers (\u003cem\u003eSNAIL1 and 2, ZEB 1 and 2, WNT1 and 4, TWIST1\u003c/em\u003e) (Supplementary Fig.\u0026nbsp;1D) than non-metastatic HCC. Well-known HCC marker GPC-3 (glypican-3) and HCP70 (Heat shock protein 70) expression was compared normal liver cell (THLE-2) and established HCC cell line (Huh7, HepG2, Hep3B) in patient-derived HCC cells (Supplementary Fig.\u0026nbsp;1E). Among the 14 patient, 3 patient-derived HCC cells were selected for current study. The patient cohort comprised four men (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2). Detailed characteristics of patient-derived hepatocellular carcinoma (HCC) and clinical features are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The non metastatic HCC cell line, YUMC-NM-H1, was isolated from the postoperative specimen of a 71-year-old woman who was unexposed to preoperative chemotherapy or radiotherapy (Supplementary Fig.\u0026nbsp;2A and E). Conversely, metastatic cell lines, YUMC-M-H1, YUMC-M-H2, and YUMC-M-H3, were isolated from specimens of three patients (53-year-old woman, 67 and 71-year-old man) with metastasis HCC at the time of surgery (Supplementary Fig.\u0026nbsp;2B\u0026ndash;D and F\u0026ndash;H); among these, two patients had peritoneal and diaphragmatic metastases. The primary tumors at the time of surgery and preoperative radiologic findings are presented in Supplementary Fig.\u0026nbsp;2. In the computed tomography (CT) findings, the yellow circle (Supplementary Fig.\u0026nbsp;2A) indicates a primary mass without metastasis (Supplementary Fig.\u0026nbsp;2E) and red circles (Supplementary Fig.\u0026nbsp;2B\u0026ndash;D) indicate metastatic HCC (Supplementary Fig.\u0026nbsp;2F\u0026ndash;H).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDifferential gene expression and signal activation between non-metastatic and metastatic HCC cells in sorafenib treatment-induced acute ER stress conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreviously, we demonstrated that acute glucose deprivation or anti-cancer drug treatment increases metabolic stress, leading to favorable subclone classification with CSC properties[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, under acute ER stress conditions, CSC-like properties and intensified induction of signaling pathways involved in survival are observed in some selectively surviving cells compared to those in their parental lineages[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To identify differences in gene expressions and signaling pathways between non-metastatic and metastatic HCC cells under anti-cancer drug-induced acute ER stress, we performed RNA sequencing-mediated transcriptome analysis. Current study, we used the patient-derived HCC cells, YUMC-NM-H1 (non-metastatic HCC) and YUMC-M-H1, -H2, and -H3 (metastatic HCC). Four tissue samples (patients were treated at the Severance Hospital, Yonsei University College of Medicine, Seoul, Korea) were obtained from two patients with divergent HCC properties (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, HCC in non-metastatic HCC was termed \u0026ldquo;YUMC-NM-H1\u0026rdquo; and that in metastatic as \u0026ldquo;YUMC-M-H1, -H2, and -H3\u0026rdquo;). Considering that gene expression is primarily dependent on metastasis, we hypothesized that metastasis involves an advanced cancer phenotype induced by epigenetic reprogramming, relative to that in non metastatic HCC cells. Therefore, metastatic HCC cell survival may involve epigenetic gene pathway induction which regulates calcium (for overburdened cytosolic free calcium regulation), peroxisome proliferator-activated receptors (PPAR; for mitochondrial metabolism), nuclear factor kappa B (NFκB; for metabolic adaptation or energy homeostasis), and Notch and Wnt signaling pathways under anti-cancer drug-treated acute ER stress conditions. NFκB is an anti-apoptotic factor known for its transcriptional role in SERCA expression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, NFκB is a pivotal regulator of mitochondrial respiration, metabolic adaptation, and energy homeostasis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Compared to non-metastatic HCC cells (YUMC-NM-H1), metastatic markers (\u003cem\u003eTYMP [Thymidine phosphorylase], EBF1 [early B cell factor 1], NDUFA4L2 [NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2], and MET [Mesenchymal epithelial transition factor]\u003c/em\u003e), multidrug resistance related gene (ABCC1 [MRP]), cancer stemness markers (\u003cem\u003ePROM1 [CD133], ALDH1A1, KRT17, KRT19, CD44, CD24 and SOX2\u003c/em\u003e) and EMT markers (\u003cem\u003eSNAIL1, SNAIL2, WNT1 WNT4, ZEB1[Zinc finger E-box-binding homeobox1], ZEB2 and TWIST1[twist family bHLH transcription factor1]\u003c/em\u003e) were significantly increased in metastatic HCC cells, YUMC-M-H1, -H2 and -H3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;H). In metastatic HCC, we concentrated on highly ranked gene modules involved in survival-related signaling pathways, including Notch, calcium, Hedgehog, and Wnt (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), particularly calcium signaling pathways which were overstimulated in metastatic HCC cells. Well-known HCC marker (GPC-3 and HCP70) and metastatic marker (TP; Thymidine phosphorylase) expression was compared normal liver cell (THLE-2) and established HCC cell line (Huh7, HepG2, Hep3B) in patient-derived HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Basal level of HCC and metastatic marker expressions were significantly increased in established HCC and patient-derived metastatic HCC than normal liver cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Basal SERCA1 and anti-apoptosis marker Bcl-2 (B cell lymphoma-2) levels (critical components involved in anti-apoptosis and calcium homeostasis)[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] were significantly higher in metastatic HCC cells than in non-metastatic HCC cells. On treatment with anti-cancer drugs such as sorafenib, SERCA1 and Bcl-2 expression notably increased in metastatic HCC cells compared to those in non-metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). SERCA2 and SERCA3 expression showed no significant differences between the two HCC cells, regardless of sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eIn summary, the calcium regulation signaling pathway or survival-related gene simulation in managing calcium regulation and SERCA are pivotal factors for survival under anti-cancer drug, sorafenib-induced metabolic stress conditions in metastatic HCC cells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetastatic HCC cell survival during prolonged anti-cancer drug treatment-stimulated metabolic stress depends on increased CaMK2α-mediated SERCA1 expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExcessive cytosolic free calcium is primarily involved in cell death[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Calcium channels, exchangers, and pumps are managed by a cytosolic-free calcium overburden and are pivotal regulators of cellular fate in physiological and pathological microenvironments[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. CaMK2α is a known as factor to increase in the nuclear translocation of NFκB which regulates of Bcl-2, SERCA and PGC1α [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We initially determined alterations in pCaMK2α, pIKKα (I kappa B kinase), Bcl-2 (anti-apoptotic marker), SERCA1, CHOP (\u003cem\u003eC/EBP homologous protein\u003c/em\u003e, ER stress marker), cleaved-caspase3 (apoptotic marker), nuclear located NFκB in the presence or absence of anti-cancer drug (sorafenib) in non-metastatic (YUMC-NM-H1) and metastatic (YUMC-M-H1, -H2 and -H3) HCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Under anti-cancer drug treatment-mediated acute ER stress conditions, metastatic HCC was evaded ER stress (CHOP) or apoptosis (cleaved-caspase3) via increase of nuclear located NFκB, Bcl-2 and SERCA1 by CaMK2α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Based on results of electrophoretic mobility shift aasy (EMSA), enhancement the nuclear translocation of NFκB (Supplementary Fig.\u0026nbsp;3A\u0026ndash;C) by stimulating CaMK2α (pCaMK2α, phosphorylated CaMK2α), which subsequently upregulated the transcription of SERCA1 and Bcl-2 in metastatic HCC cells compared to that in non-metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Supplementary Fig.\u0026nbsp;3A\u0026ndash;C). Consequently, we evaluated the causal relationship between elevated SERCA1 levels and CaMK2α by CaMK2α knockdown in metastatic HCC (YUMC-M-H1, -H2, and -H3 small interfering [si]-\u003cem\u003eCaMK2α\u003c/em\u003e) with sorafenib (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;E). Immunoblot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and cell viability assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) indicated that \u003cem\u003eCaMK2α\u003c/em\u003e knockdown in metastatic HCC cells using siRNAs downregulated cell viability and decrease of pIKKα, Bcl-2 and SERCA1 levels through inhibition of nuclear translocated NFκB under sorafenib treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C). Consequently, \u003cem\u003eCaMK2α\u003c/em\u003e knockdown in metastatic HCC cells significantly increased the levels of apoptotic markers CHOP and cleaved caspase 3, while considerably decreasing those of pCaMK2α, pIKKα, Bcl-2, SERCA1, and nuclear translocated NFκB under acute ER stress conditions by sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Viability assays showed the dose-dependent restriction of prolonged survival with sorafenib in downregulated CaMK2α metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, left; YUMC-M-H1, middle; YUMC-M-H2, right; YUMC-M-H3). Immunofluorescence assays of \u003cem\u003eCaMK2α\u003c/em\u003e knockdown in metastatic HCC cells showed that the nuclear translocation of NFκB was considerably inhibited by \u003cem\u003eCaMK2α\u003c/em\u003e knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Microspectrofluorimetry revealed that the \u003cem\u003eCaMK2α\u003c/em\u003e knockdown-mediated inhibition of SERCA1 and Bcl-2 influenced intracellular calcium levels in metastatic HCC cells following sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Cytosolic free calcium levels were measured in response to high potassium depolarization, which triggered an increase in the levels of cytosolic free calcium; its clearance was estimated in the presence or absence of sorafenib based on separate calcium fluxes. Cytosolic free calcium levels were restored to the basal levels in the control and si-scrambled group after sorafenib each treatment in metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, top; YUMC-M-H1, middle; YUMC-M-H2 and bottom; YUMC-M-H3). However, this restoration was interrupted by CaMK2α knockdown via si\u003cem\u003e-CaMK2α\u003c/em\u003e in metastatic HCC cells.\u003c/p\u003e\u003cp\u003eIn summary, metastatic HCC cells can ameliorate calcium-mediated apoptosis under anti-cancer drug treatment by increasing SERCA1 levels through inducing nuclear NFκB translocation via CaMK2α, which is a pivotal transcriptional contributor to SERCA1 and Bcl-2 upregulation.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eCandidate 56 and 62 identification as SERCA1 target-specific inhibitors using molecular docking simulation and structure modeling\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe hypothesized and demonstrated that metastatic HCC cells and functional SERCA1 inhibition could be a practical clinical approach for patients with metastatic HCC. We identified novel, small SERCA1-specific binding molecules as well as the possibility of pharmacophore-binding modes. Consequently, we identified two novel small molecules, candidate 56 and 62, which showed pharmacophoric high SERCA1-specific binding affinity, resulting in critical functional suppression of SERCA1. An evolutionary chemical binding similarity (ECBS) program was used to elucidate the inhibitory mechanisms of candidate 56 and 62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). ECBS method using a categorization similarity learning framework defined with paired chemical data and target\u0026rsquo;s evolutionary relationship. The ECBS method is designed to encode molecular features enriched in evolutionarily conserved chemical-target binding relationships, and formulated by the likelihood of chemical compounds binding to identical targets[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To recognize the molecular interactions of candidate 56 and 62, we carried out blind docking simulations, followed by local docking refinement and energy minimization. A blind docking simulation was performed using the human SERCA1 structural model to predict the ligand-binding site and molecular interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and G). A similarity search using FoldSeek[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] server revealed that the structure of human SERCA1 is highly similar to that of rabbit SERCA1 (PDB ID 6YSO, UniProt ID P04191), with a sequence identity of 95.3% and root mean square deviation of 1.89 \u0026Aring; (TM-Score 0.971), indicating significant consistency between them. Docking complex models suggested the potential binding of candidate 56 and 62 to the cavity between the n and p domains in the cytoplasmic region of SERCA1, overlapping with the binding site of the ATP derivative (PubChem CID 644358) found in the rabbit SERCA1 structure[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The docking scores, obtained through all-atom minimization with Rosetta, were \u0026minus;\u0026thinsp;14.3 and \u0026minus;\u0026thinsp;13.4 (in Rosetta Energy Unit) for candidate 56 and 62, respectively. Despite their similar binding scores and chemical structures, candidate 56 and 62 exhibited contrasting binding orientations in the docking models, presumably due to their distinct 3D conformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). In candidate 56, the aromatic ring with a chlorine atom was directed towards F487 and M494; whereas in candidate 62, it interacted with P518 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Notably, F487 consistently participated in pi-pi interactions with the aromatic rings of both compounds. Additionally, R560 showed a polar interaction only with candidate 62, presumably contributing to its binding specificity, along with additional interactions with L562 and T441. In contrast, A517 and K492 cells exhibited more hydrophobic interactions with candidate 56. Pharmacophore models based on the receptor\u0026ndash;ligand interactions are constructed to highlight the essential interactions within the ligands (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These ligand\u0026ndash;protein interactions observed in the docking models have the potential to offer valuable insights into the molecular basis of their activity, aiding efficient molecule design in the future.\u003c/p\u003e\u003cp\u003eThese protein-ligand interactions, as suggested by the docking models, have the potential to elucidate the molecular basis of the ligand activity. However, experimental validation is still necessary in future studies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCandidate 56 and 62 increase of restraint the survival of metastatic HCC cells through failed to revert to basal levels after cytosolic free calcium spike\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe conducted cell viability assays to assess the anti-cancer impact of the novel small molecules and SERCA1-specific inhibitors, candidate 56 and 62. The results showed that non-metastatic HCC cells exhibited considerably reduced viability in a dose-dependent manner following sorafenib treatment, regardless of whether it was used in combination with SERCA inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Treatment with candidate 56 or 62 alone had a marginal anti-cancer effect on non-metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Moreover, the viability of metastatic HCC cells was not significantly affected by the SERCA inhibitors or sorafenib treatment alone. In contrast, novel SERCA1-specific inhibitor candidate 56 and 62 used in combination with sorafenib remarkably reduced metastatic HCC cell viability in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;D). SERCA is a pivotal player and therapeutic target in the regulation of cytosolic calcium overburden in cancer[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The anti-cancer effect of candidate 56 and 62 via the specific functional inhibition of SERCA1 was determined using microspectrofluorometry based on the differences in intracellular calcium levels in patient-derived non-metastatic and metastatic HCC cells treated with sorafenib alone or with SERCA inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;H). Free cytosolic calcium levels were measured in the presence of high-potassium depolarization and were estimated alone or combined sorafenib treatment with SERCA inhibitors (Thapsigargin; positive control, candidate 56, and candidate 62). Cytosolic free calcium levels in non-metastatic HCC cells failed to revert to basal levels following sorafenib treatment, regardless of whether it was used in combination with SERCA inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Sorafenib treatment alone in metastatic HCC cells, the cytosolic free calcium levels revert to the basal levels. However, when SERCA1-specific inhibitors candidate 56 and 62 were combined with each, cytosolic free calcium failed to revert to basal levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;H). There were no significant changes in the levels of cytosolic free calcium on treatment with sorafenib or SERCA inhibitors alone in metastatic HCC cells.\u003c/p\u003e\u003cp\u003eThese variations in cytosolic free calcium levels between patient-derived non-metastatic and metastatic HCC cells may be directly related to SERCA. To assess whether the prolonged survival of metastatic HCC cells on sorafenib treatment was indeed associated with only SERCA1 and not sodium-calcium exchangers (NCX), not calcium ion channels, not plasma membrane calcium ATPase (PMCA) we performed cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;D) and immunoblot assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) on treatment with NCX inhibitor (KB-R7943), calcium channel blockers (bepridil, verapamil or nifedipine), plasma membrane calcium PMCA inhibitor (caloxin2a1), and SERCA inhibitors in combination with sorafenib in non-metastatic and metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;E). In non-metastatic HCC cells, no significant changes were observed on treatment with either agent with csorafenib compared to that with sorafenib treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In metastatic HCC cells, sorafenib treatment alone or combination treatment with NCX inhibitor, calcium channel blockers, PMCA inhibitor did not significantly influence survival, whereas combination treatment with SERCA inhibitors were failed to survive dose-dependently (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C, and D). Results of the immunoblot assay indicated that CHOP (an ER stress marker) levels increased significantly on combining SERCA inhibitors and sorafenib treatment in metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, top; right, bottom; left and right). However, in non-metastatic HCC cells, CHOP levels significantly increased in the presence of sorafenib regardless of treatment with an NCX inhibitor, calcium channel blocker, SERCA inhibitor, or PMCA inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, top; left). To verify the role of candidate 56 and 62 as SERCA1-specific inhibitors, we assessed the anti-cancer effects of candidate 56 or 62 when used in combination with each anti-cancer drug (sorafenib, 5-Fluorouracil [5-FU] and gemcitabine) in other metastatic HCC cells (YUMC-M-H8; patient-derived HCC cells were isolated from metastatic tissue of a patient after 5FU therapy, YUMC-M-H12; patient-derived HCC cells were isolated from metastatic tissue of a patient after gemcitabine therapy) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-I). mRNA quantification of metastatic HCC cells compare to non-metastatic HCC cells under each anti-cancer drug treated conditions showed increase in SERCA1 levels predominantly in YUMC-M-H1, -H2, and -H3; however, YUMC-M-H8 and YUMC-M-H12 showed significantly increased levels of the SERCA2 or SERCA3 isoforms respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Protein quantification at the basal or anti-cancer drug (sorafenib, 5-FU and gemcitabine)-treated conditions using immunoblot assay further proved that the metastatic HCC cells, YUMC-M-H1, -H2, and -H3 significantly expressed SERCA1, whereas other metastatic HCC cells, YUMC-M-H8 and -H12 (patient-derived HCC cells, metastasis ocurred after 5-FU or gemcitabine therapy) significantly increased the SERCA2 and SERCA3 isoforms, respectively under anti-cancer drug treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). ER stress marker, CHOP levels were not significantly different in metastatic HCC cells regardless of the anti-cancer drug used. However, non-metastatic HCC cells showed significantly increased CHOP levels on sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In cell viability assays, combination treatment with the novel SERCA1 isoform-specific inhibitors (candidate 56 and 62) and anti-cancer drugs (sorafenib, 5-FU and gemcitabine), resulted in dominantly expressed SERCA1 (YUMC-M-H1, -H2, and -H3), SERCA2 (YUMC-M-H8), and SERCA3 (YUMC-M-H12), respectively, in the metastatic HCC cell line. Consequently, cell viability of SERCA1 dominantly expressed metastatic HCC cells was suppressed by SERCA1 isoform-specific inhibitors (candidate 56 and 62) with anti-cancer drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, left), whereas SERCA2 or SERCA3 dominantly expressed HCC was no significantly influenced candidate 56 and 62 with anti-cancer drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eH, right). Cell viability significantly decreased whereas CHOP levels increased in non-metastatic HCC cells on sorafenib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). However, metastatic HCC cells (YUMC-M-H1, -H2, -H3, -H8, and -H12) did not significantly influence cell viability or ER stress under anti-cancer drug (sorafenib, 5-FU and gemcitabine) alone treated conditions. These cells showed significantly decreased cell viability and increased CHOP levels following treatment with the SERCA1 isoform-specific inhibitors, candidate 56 and 62 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). However, in SERCA2 and SERCA3 dominantly expressed HCC cells, YUMC-M-H8 and -H12, respectively, showed no significant difference in cell viability and increase in ER stress on combined treatment with anti-cancer drugs (5FU or gemcitabine) and candidate 56 or 62. Therefore, the novel SERCA inhibitors candidate 56 and 62 could be considered as SERCA1-specific inhibitors.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThese findings imply that SERCA1 regulates survival prolongation in metastatic HCC cells during chemotherapy treatment by mediating overloaded cytosolic free calcium levels through new SERCA1 isoform-specific inhibitors, candidate 56 and 62.\u003c/p\u003e\u003cp\u003e\u003cb\u003eA novel therapeutic approach for metastatic HCC through SERCA1-specific inhibitors (novel small molecules, candidate 56 and 62) in a patient-derived metastatic HCC cell mouse xenograft model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe evaluated the anti-cancer effects of candidate 56 and 62 \u003cem\u003ein vivo\u003c/em\u003e using a mouse xenograft tumor model with SERCA1, which predominantly increased patient-derived metastatic cells. We induced acute ER stress by treatment with sorafenib alone or with SERCA inhibitors. The dose of candidate 56 and 62 in xenograft model was selected in a dose-dependent manner following candidate 56 and 62 treatment with sorafenib (Supplementary Fig.\u0026nbsp;4A\u0026ndash;C). In the xenograft model with non-metastatic HCC cells, conspicuous tumor shrinkage increased due to sorafenib treatment regardless of SERCA inhibitor combination treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, top). In the metastatic HCC xenograft model, sorafenib treatment alone did not increase tumor shrinkage; however, combinatorial treatment with sorafenib and candidate 56 or 62, novel SERCA1 isoform-specific inhibitors resulted in remarkable tumor shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;D, top). The excised tumors were similar in terms of tumor volume, which declined considerably with sorafenib treatment, regardless of the presence of SERCA inhibitors in the non-metastatic HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, middle). By comparison, dissected tumor weight in metastatic HCC cells was not meaningful influenced by each sorafenib alone, whereas combinatorial strategy to sorafenib with candidate 56 or 62 leaded a prominent decrease in tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;D, middle). All the inhibitory agents, administered alone or in combination, did not significantly influence the whole body weight of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D, bottom). Further, morein the change of survival rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and whole body weight (Supplementary Fig.\u0026nbsp;4D) were measured under treatment with thapsigargin, candidate 56, or 62 alone in normal mice (not xenograft model) and compared with a non-treated group for 29 days. In normal mice, candidate 56 or 62 treatment alone showed no significant difference in whole body weight or death ratio compared with the group treated with thapsigargin alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;4D). Tumor lysate-based immunoblot assay of non-metastatic HCC cells revealed that SERCA1 expression did not significantly change; however, CHOP expression was significantly induced with sorafenib treatment alone or in combination with SERCA inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). In contrast, metastatic HCC cells showed high SERCA1 expression following treatment with sorafenib alone or in combination with SERCA inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026ndash;I). Moreover, the sorafenib-induced overexpression of CHOP was significantly alleviated by an increase in SERCA1. However, the functional inhibition of SERCA1 using SERCA1-specific inhibitors, candidate 56 and 62 increased in acute ER stress (CHOP induction) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026ndash;I). Candidate 56 and 62, administered alone did not significantly influence the hepatic injury of mice compare than carbon tetrachloride alone treatment (CCl\u003csub\u003e4\u003c/sub\u003e, positive control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Moreover, candidate 56 and 62-mediated cardiac injury was no significantly induced in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eO\u0026ndash;R).\u003c/p\u003e\u003cp\u003eThe increase in SERCA1 in metastatic HCC represents a targetable mechanism to overcome resistance. Consequently, in metastatic HCC, CaMK2α-mediated SERCA1 increase becomes a pivotal factor for survival under acute ER stress. These new small molecules, the SERCA1-specific inhibitors candidate 56 and 62, present a promising therapeutic approach for unmet medical needs without causing side effects and could potentially treat patients with anti-cancer drug-resistant-mediated metastatic and recurrent cancer at lower doses than those required for individual anti-cancer drug use.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe advancement of anti-cancer drugs has led to numerous studies demonstrating the benefits of preoperative treatments in enhancing survival rates post-surgery[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Despite this progress, there remains a lack of established therapeutic options as standard neoadjuvant or adjuvant treatments for HCC[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], leading to a continual increase in unmet medical needs. A significant portion of these unmet needs is attributed to the recurrence and metastasis mediated by anti-cancer drug resistance[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, targeting anti-cancer drug resistant mediated-metastatic cancer cells remains a significant challenge in cancer treatment and research[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The mechanism of cancer metastasis in patients with cancer differs significantly for each cancer, owing to specific attributes that negatively affect cancer therapy and eventually cause cancer progression and death[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Consequently, a clinical solution for anti-cancer drug resistant-mediated metastatic cancer is an unattainable target. In the current study, we postulated a therapeutic framework for metastatic cancer based on the results of a prototypical model experiment. The ultimate goal of anti-cancer drugs is to induce cancer cell death. However, anti-cancer drug resistant-mediated metastatic cancer cells evade death through epigenetic reprogramming, which upregulates numerous target genes[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Among the various mechanisms of evading cell death, refractory cancer could be resistant to ER stress by evading excess cytosolic free calcium-mediated apoptosis under anti-cancer drug treatment or glucose starvation metabolic stress[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and phenocopy cytotoxic chemotherapy-resistant cancers[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Conventional anti-cancer drugs target the increase in cytosolic free calcium, which causes calcium-mediated apoptosis in anti-cancer drug-sensitive cancer cells[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Management of excess cytosolic free calcium is critical in apoptosis-resistant cancer cells and is facilitated by the calcium pump[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In refractory cancers such as anti-cancer drug-resistant-mediated cancer cells many target genes are upregulated for survival under acute ER stress conditions[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. SERCA is a critical target protein that supports low levels of resting cytosolic free calcium in cancer cells[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, enhanced SERCA expression is associated with poor outcomes in cancer, since it protects cells from excess cytosolic free calcium-mediated apoptosis[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe results of this study showed that, among the SERCA isoforms, SERCA1 levels predominantly increased in patient-derived metastatic HCC cells and further identified two novel small molecules, candidate 56 and 62, which were SERCA1-structure based specific inhibitors. mRNA sequencing (mRNA-Seq) analysis was based on the ECBS program, pharmacophore, and docking-based sequential virtual screening for identifying novel SERCA1-specific inhibitors, to devise a therapeutic strategy for refractory cancer. mRNA-Seq and immunoblot analyses revealed higher SERCA1 expression in metastatic HCC cells than that in non-metastatic HCC cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that Calcium and Notch signaling pathways ranked high among the top 10 signaling pathways in patient-derived metastatic HCC cells compared to those in patient-derived non-metastatic HCC cells. The correlation between Calcium and Notch signaling pathways is well-established[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], and Notch signaling is regulated by SERCA suppression[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Consequently, we identified SERCA isoforms and excess cytosolic free calcium-mediated apoptosis as key players in numerous upregulated target genes. The present study findings revealed that enhanced SERCA1 transcription through NFκB nuclear translocation by CaMK2α plays a pivotal role in evading excess cytosolic free calcium mediated apoptosis under acute ER stress conditions in anti-cancer drug (sorafenib)-induced cytotoxic stress-resistant HCC cells. Increase in SERCA1 levels by CaMK2α enhances the restoration of excess cytosolic free calcium, which mainly contributes to anti-cancer drug-resistance-mediated metastasis to anti-cancer drug treatment-elevated acute ER stress. Increase in SERCA1 expression by CaMK2α restrains excess calcium-dependent apoptosis.\u003c/p\u003e\u003cp\u003eNotably, the current study proposes a reliable therapeutic approach for patient-derived metastatic HCC by identifying novel SERCA1-specific inhibitors, candidate 56 and 62. The present study findings validate the role of candidate 56 and 62, which could be prevent cardiac injury. SERCA isoforms are well-known key regulators of cardiac muscle, and therapeutic approaches to these SERCA inhibitors are unavoidably involved in cardiac dysfunction. Consequently, patients with SERCA-dependent anti-cancer drug-resistant-mediated metastatic cancer may also be concerned about cardiac dysfunction. However, identification and validation of SERCA isoform-specific inhibitors, as in the current study, may potentially decrease cardiac dysfunction. Findings of current study might be favorable to founding prospective, reasonable clinical approaches in patients with refractory HCC to advance efficacious theraphy. In a clinical relevance, these outcomes of current study impart significant implications for the development of novel combinatorial strategies and the discovery of new anti-cancer candidates that target a specific vulnerability of malignant cells, such as drug resistant-mediated etastatic cancer cells. However, several further studies were required to establish the current clinical approach. Furthermore, more study is needed owing to the limitations of several patient results. To overcome these limitations, several studies are on going on various types of patient-derived anti-cancer drug-resistant-mediated metastatic cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cb\u003eSERCA\u003c/b\u003e sarco/endoplasmic reticulum calcium ATPase\u003c/p\u003e\u003cp\u003e\u003cb\u003eCaMK2α\u003c/b\u003e Calcium/calmodulin-dependent protein kinase alpha\u003c/p\u003e\u003cp\u003e\u003cb\u003eNFκB\u003c/b\u003e nuclear factor kappa B\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA-Seq\u003c/b\u003e RNA sequencing\u003c/p\u003e\u003cp\u003e\u003cb\u003eqRT-PCR\u003c/b\u003e quantitative reverse transcription PCR\u003c/p\u003e\u003cp\u003e\u003cb\u003eCSCs\u003c/b\u003e cancer stem cells\u003c/p\u003e\u003cp\u003e\u003cb\u003eER\u003c/b\u003e endoplasmic reticulum\u003c/p\u003e\u003cp\u003e\u003cb\u003esiRNA\u003c/b\u003e small interfering RNA\u003c/p\u003e\u003cp\u003e\u003cb\u003eRPMI-1640\u003c/b\u003e Rosewell Park Memorial Institute-1640\u003c/p\u003e\u003cp\u003e\u003cb\u003eFBS\u003c/b\u003e Fetal Bovine Serum\u003c/p\u003e\u003cp\u003e\u003cb\u003eYUMC\u003c/b\u003e Yonsei University Medical Center\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank proffesor Chan Wung Kim (CKP Therapeutics, Inc., Massachusetts Medical Device Development Center, 110 Canal Street, Lowell MA 01852, USA) for supporting this research. Particulary we extend our sincere gratitude to Mung-Kun Park and Hyun Joo Hwang for their invaluable support in this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJHL, KP, KHC, SMK, KCP and JHC primarily conducted the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies and contributed to manuscript drafting. SMK and KCP isolated the patient-derived drug-resistant cancer cells. JMK, YLJ, KP and KHC performed statistical analyses. JHL, KP, KHC, SMK, KCP and JHC contributed to manuscript drafting and study design. JHL, SMK, KHC, KCP and JHC conceptualized the study, designed experiments, prepared the manuscript, and finalized it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received support from a grant from the KHIDI, funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (HI18C1188), the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2017R1D1A1B03029716), and CKP Therapeutics, Inc. (grant numbers: 2021-31-1118), the \u0026nbsp;Ministry \u0026nbsp;of \u0026nbsp; Trade, Industry \u0026amp; Energy (MOTIE), \u0026nbsp; Korea Planning \u0026amp; Evaluation Institute of Industrial Technology (KEIT) through the Encouragement Program \u0026nbsp; for \u0026nbsp;Technology Development (Project \u0026nbsp;NO: \u0026nbsp;RS-2024-00410585).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research protocol was approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine (IRB Protocol: 3-2022-0331). Cell samples were obtained from patients at the Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea. This study was performed in accordance with the Declaration of Helsinki. The requirement for obtaining informed consent was waived owing to the retrospective nature of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files, or available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlqahtani A, Khan Z, Alloghbi A, Said Ahmed TS, Ashraf M, Hammouda DM. 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Front Immunol. 2022;13:832159.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePagliaro L, Marchesini M, Roti G. Targeting oncogenic Notch signaling with SERCA inhibitors. J Hematol Oncol. 2021;14(1):8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cdiv class=\"SimplePara\"\u003eProperties and clinical features of patients. Patient-derived Hepatocellular carcinoma cells were isolated from these patient specimen.\u003c/div\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eYUMC-NM-H1\u003c/div\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eYUMC-M-H1\u003c/div\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eYUMC-M-H2\u003c/div\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eYUMC-M-H3\u003c/div\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eAge at Diagnosis\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003e71\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003e53\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003e67\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003e71\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eGender\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eFemale\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eFemale\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eMale\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eMale\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003ePrimary\u003c/span\u003e\u003c/div\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eDisease Site\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eLiver\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eLiver\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eLiver\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eLiver\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eStage\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eT1bN0M0\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eT3N0M1\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eT1N1M1\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eT2N1M1\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003ePrimary\u003c/span\u003e\u003c/div\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003ePathology\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eHepatocellular carcinoma\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eHepatocellular carcinoma (Metastasis after sorafenib treatment)\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eHepatocellular carcinoma (Metastasis after sorafenib treatment)\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eHepatocellular carcinoma (Metastasis after sorafenib treatment)\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eClassification of specimen used for culture\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eFresh tumor\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eFresh tumor\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eFresh tumor\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eFresh tumor\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eObtained from\u003c/span\u003e\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cdiv class=\"SimplePara\"\u003eSeverance Hospital, Seoul, Korea\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cdiv class=\"SimplePara\"\u003eSeverance Hospital, Seoul, Korea\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cdiv class=\"SimplePara\"\u003eSeverance Hospital, Seoul, Korea\u003c/div\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cdiv class=\"SimplePara\"\u003eSeverance Hospital, Seoul, Korea\u003c/div\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003cbr/\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"patient-derived metastatic HCC, sarcoplasmic/endoplasmic reticulum calcium ATPase, calcium/calmodulin-dependent protein kinase 2 alpha","lastPublishedDoi":"10.21203/rs.3.rs-7382767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7382767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eRefractory hepatocellular carcinoma (HCC) perpetuates metastasis or recurrence through anti-cancer drug resistance, necessitating more effective and reliable therapeutic strategies.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe propose a new therapeutic approach involving the discovery of novel small molecules through target identification and validation in a patient-derived metastatic HCC model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe showed that calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α)-mediated enhancement of sarco/endoplasmic reticulum (ER) calcium ATPase 1 (SERCA1) expression level was pivotal events under anti-cancer drug treated conditions in patient-derived metastatic HCC cells. Increased SERCA1 was regulates to overloaded free calcium. SERCA is widely recognized as a key regulator of cytosolic free calcium under severe ER stress conditions. However, a cardiac dysfunction was inevitable in vivo because of non-specific inhibition of SERCA isoforms by conventional SERCA inhibitors. Based on the molecular structure of SERCA1, we discovered and synthesized two SERCA1-specific inhibitors, candidate 56 and 62. These compounds significantly reduced tumor size in the metastatic HCC xenograft tumor model without cardiac contractile dysfunction.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study first showed survival mechanism of patient-derived metastatic HCC cell, and propose a new therapeutic approach by the new small molecules, candidate 56 and 62, which are SERCA1 isoform-specific inhibitors without cardiac dysfunction by SERCA1 selectively inhibition.\u003c/p\u003e","manuscriptTitle":"Discveory of SERCA1 specific small molecule inhibotirs based on the survival mechanisms of metastatic hepatocellular carcinoma cells dependent on CaMK2α-Mediated SERCA1 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 11:02:56","doi":"10.21203/rs.3.rs-7382767/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"acd6c315-3b46-4ccb-9672-d1f47d3271eb","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-17T11:02:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 11:02:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7382767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7382767","identity":"rs-7382767","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}